Search

Gay men are more likely than heterosexual men to experience social pressure based on body weight, shape, and muscularity, which may drive disparities in body image concerns and eating disorders. Utilizing a sample of 1723 gay men living in the United States, the present study examined whether sociodemographic factors (used as proxies for status and sexual capital) and frequency of attending gay-specific establishments or gatherings (community involvement) were associated with gay men's experiences of negative or discriminatory pressures based on body size and shape specifically from other gay men (intraminority body stigma). Experiences of intraminority body stigma were significantly more common among gay men who identified as higher-weight (r = 0.28), less masculine (r = -0.21), less wealthy (r = -0.21), younger (r = -0.21), or people of color (ds = 0.25-0.28). Furthermore, indicators of low status and sexual capital were indirectly associated with less frequent community involvement via more frequent experiences of intraminority body stigma. In addition to frequency, the valence of interactions between gay men should be considered when assessing body image and eating disorder risk in this population. Future research is encouraged to examine intraminority body stigma as an intersectional source of intraminority stress to inform prevention and treatment efforts for gay men., Competing Interests: Declaration of Competing Interest We have no known conflict of interest to disclose., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

There is currently a lack of measures testing intraminority stress within gay men. Therefore, the current study sought to develop and psychometrically test the Gay-Specific Intraminority Stigma Inventory (G-SISI). Based on a content review of the literature and a panel of experts, a pool of items assessing gay men's perceived exposure to a range of discriminatory attitudes from other gay men was generated. Utilizing a randomly split sample of 1723 gay men between the ages of 19 and 79 years, an exploratory factor analysis was first performed ( n = 861). The remaining unexamined data were then used to conduct a confirmatory factor analysis ( n = 862). The results support a six-factor model: (1) Age Stigma, (2) Socioeconomic Stigma, (3) Gay Non-Conformity Stigma, (4) Racial Stigma, (5) Gender Expression Stigma, and (6) Body Stigma. Cronbach's alpha for the total scale was 0.90 and for the subscales ranged from 0.60 to 0.85. Sociodemographic factors and measures of community involvement were differentially associated with the G-SISI subscales, providing evidence of construct validity. The findings demonstrate initial support for the dimensionality and validity of the G-SISI, which targets modifiable factors (e.g., identity-based stigma) that may increase stress and reduce community coping resources among gay men with diverse identities.

Raccoon rabies is endemic in the eastern U.S.; however, an epizootic had not been confirmed on Long Island, New York until 2004. An oral rabies vaccination (ORV) program was initiated soon after the first rabies-positive raccoon was discovered, and continued until raccoon rabies was eliminated from the vaccination zone. The cost-effectiveness and economic impact of this rabies control program were unknown. A public health surveillance data set was evaluated following the ORV program on Long Island, and is used here as a case study in the health economics of rabies prevention and control efforts. A benefit-cost analysis was performed to determine the cost-effectiveness of the program, and a regional economic model was used to estimate the macroeconomic impacts of raccoon rabies elimination to New York State. The cost of the program, approximately $2.6 million, was recovered within eight years by reducing costs associated with post-exposure prophylaxis (PEP) and veterinary diagnostic testing of rabies suspect animals. By 2019, the State of New York is projected to benefit from the ORV program by almost $27 million. The benefit-cost ratio will reach 1.71 in 2019, meaning that for every dollar spent on the program $1.71 will be saved. Regional economic modeling estimated employment growth of over 100 jobs and a Gross Domestic Product (GDP) increase of $9.2 million through 2019. This analysis suggests that baiting to eliminate rabies in a geographically constrained area can provide positive economic returns., Competing Interests: JLM is an employee of Merial Inc., who funded the study and whose vaccine was used in the rabies control program in question in this paper. This study considers the costs of a rabies control program compared with the benefits of rabies elimination. As the efficacy of the vaccine is not analyzed in our study, we do not feel there is any conflict of interest.

RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.

Necrosis is a primary form of cell death in a variety of human pathologies. The deleterious nature of necrosis, including its propensity to promote inflammation, and the relative lack of the cells displaying necrotic morphology under physiologic settings, such as during development, have contributed to the notion that necrosis represents a form of pathologic stress-induced nonspecific cell lysis. However, this notion has been challenged in recent years by the discovery of a highly regulated form of necrosis, termed regulated necrosis or necroptosis. Necroptosis is now recognized by the work of multiple labs, as an important, drug-targetable contributor to necrotic injury in many pathologies, including ischemia-reperfusion injuries (heart, brain, kidney, liver), brain trauma, eye diseases, and acute inflammatory conditions. In this review, we describe the methods to analyze cellular necroptosis and activity of its key mediator, RIP1 kinase., (© 2014 Elsevier Inc. All rights reserved.)

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3mg of highly purified RIP1 kinase was typically obtained from a 1L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Necroptosis is a novel form of regulated non-apoptotic cell death, which displays morphological features of necrosis. The kinase activity of receptor-interacting protein kinase 1 (RIP1) is a critical component in signaling for necroptosis. The development of assays to evaluate RIP1 kinase activity is important in the further development of existing and novel inhibitors of necroptosis. Here, we describe RIP1 protein expression and purification from mammalian and insect cells as well as two in vitro kinase assays to detect RIP1 kinase activity and inhibition.

Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, "closed" state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more "open", has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.

Purpose: To evaluate accuracy of the referring diagnoses of retinoblastoma to a tertiary cancer referral center., Methods: Retrospective chart review of 352 retinoblastoma-related patients seen by the ophthalmic oncology service during a 4-year period from January 1, 2004 to October 21, 2008. Of these, 111 were referred with a suspicion of new retinoblastoma and were included in the study. Fundus photographs, gender, family history of retinoblastoma, initial symptoms, age, initial and referring physicians' specialty (eg, pediatrician, general ophthalmologist, retinal specialist) and their suspected diagnoses were recorded. The main outcome measure was accuracy of diagnosis given by referring providers., Results: Of 111 patients, 62% had retinoblastoma and 38% did not. Persistent fetal vasculature (PFV) and Coats' Disease were the most common simulating lesions accounting for 31% and 29% of the simulating lesions respectively. Other simulating lesions included infrequent cases of rare conditions such as primary ocular teratoma, a retinal pigment epithelial tumor, and astrocytic hamartoma., Conclusions: Retinoblastoma continues to present a diagnostic dilemma. There has been limited improvement in the rate of correct diagnosis in the United States in the last 15 years. There has however been a change in the composition of misdiagnosed lesions with rare conditions accounting for more than 1/3 of cases. Attention to age, family history, laterality and presenting signs such as globe size can aid diagnosis of retinoblastoma.

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future., ((c) 2007 Wiley Periodicals, Inc.)

We describe a clinical scenario in which a child was treated for glaucoma and aqueous misdirection with surgical placement of a tube shunt. The child then suffered extrusion of the tube through the cornea, a rare complication of such procedures. This complication has only been seen in children, and thus, young patients with tube shunts need careful and frequent monitoring, especially during the first year of the post tube shunt period to ensure that they have not developed this complication.

Twenty-four adult striped skunks (Mephitis mephitis) were administered the raccoon product formulation of Rabies Vaccine, Live Vaccinia-Vectored (Raboral V-RG, Merial Limited, Athens, Georgia, USA), either by oral instillation or in vaccine-filled coated sachets either as single or multiple doses. A control group remained unvaccinated. Twenty-three of the skunks were challenged 116 days postvaccination with rabies virus (skunk isolate). Six of six naive skunks succumbed to challenge. Four of six skunks that received the vaccine by oral instillation survived challenge. The skunks that did not survive failed to seroconvert following vaccination. None of the skunks that accepted multiple doses of the vaccine offered in coated sachets survived challenge, nor were rabies virus-neutralizing antibodies (VNAs) detected in the sera. Likewise, none of the five skunks ingesting a single sachet developed VNA against rabies. However, in this group one skunk did survive rabies challenge. This preliminary study showed that the vaccinia-vectored oral rabies vaccine Raboral V-RG, as formulated for use in raccoons, is capable of protecting a percentage of skunks against rabies. However, although the fishmeal-coated sachets were readily consumed, subsequent challenge of these animals revealed poor vaccine delivery efficiency.

At low concentrations, hydrogen peroxide (H(2)O(2)) is a positive endogenous regulator of mammalian cell proliferation and survival; however, the signal transduction pathways involved in these processes are poorly understood. In primary human endothelial cells, low concentrations of H(2)O(2) stimulated the rapid phosphorylation of the acidic C-terminal domain (ACD) of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear restricted pre-mRNA-binding protein, at Ser(240) and at Ser(225)-Ser(228). A kinase activity was identified in mouse liver that phosphorylates the ACD of hnRNP-C at Ser(240) and at two sites at Ser(225)-Ser(228). The kinase was purified and identified by tandem mass spectrometry as protein kinase CK1alpha (formerly casein kinase 1alpha). Protein kinase CK1alpha immunoprecipitated from primary human endothelial cell nuclei also phosphorylated the ACD of hnRNP-C at these positions. Pretreatment of endothelial cells with the protein kinase CK1-specific inhibitor IC261 prevented the H(2)O(2)-stimulated phosphorylation of hnRNP-C. Utilizing phosphoserine-mimicking Ser-to-Glu point mutations, the effects of phosphorylation on hnRNP-C function were investigated by quantitative equilibrium fluorescence RNA binding analyses. Wild-type hnRNP-C1 and hnRNP-C1 modified at the basal sites of phosphorylation (S247E and S286E) both avidly bound RNA with similar binding constants. In contrast, hnRNP-C1 that was also modified at the CK1alpha phosphorylation sites exhibited a 14-500-fold decrease in binding affinity, demonstrating that CK1alpha-mediated phosphorylation modulates the mRNA binding ability of hnRNP-C.

Retroviral assembly is driven by multiple interactions mediated by the Gag polyprotein, the main structural component of the forming viral shell. Critical determinants of Gag oligomerization are contained within the C-terminal domain (CTD) of the capsid protein, which also harbors a conserved sequence motif, the major homology region (MHR), in the otherwise highly variable Gag. An unexpected clue about the MHR function in retroviral assembly emerges from the structure of the zinc finger-associated SCAN domain we describe here. The SCAN dimer adopts a fold almost identical to that of the retroviral capsid CTD but uses an entirely different dimerization interface caused by swapping the MHR-like element between the monomers. Mutations in retroviral capsid proteins and functional data suggest that a SCAN-like MHR-swapped CTD dimer forms during immature particle assembly. In the SCAN-like dimer, the MHR contributes the major part of the large intertwined dimer interface explaining its functional significance.

Fish acquire protective immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection or inoculation with I. multifiliis immobilization antigens (i-antigens). In both cases, parasite-immobilizing antibodies have been identified in sera and mucosal secretions. To investigate the kinetics of this immune response, antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in the sera and cutaneous mucus of channel catfish (Ictalurus punctatus) that were either infected with parasites or given a single injection of purified i-antigen (5.0 microg/fish) in Freund's incomplete adjuvant. At 5 weeks, infected and inoculated fish had a mean serum (1:80 dilution) antibody absorbance (A405) value of 0.54 +/- 0.17 and 0.35 +/- 0.03, respectively, which were significantly higher (alpha = 0.05) than the pretreatment serum (1:80 dilution) antibody absorbance value of 0.24 +/- 0.05. At 14 weeks, mean serum (1:80 dilution) ELISA absorbance values in the teo groups of fish increased to 0.79 +/- 0.30 and 0.71 +/- 0.24, respectively. In both groups of fish, antibody levels in cutaneous mucus (undiluted) were much lower than those in sera. Infected fish had detectable mucus (undiluted) antibody levels from 3 to 9 weeks, with the highest mean value (0.30 +/- 0.07) occurring at 7 weeks. Although individual inoculated fish produced serum antibody absorbance values comparable to those seen in infected fish, the mean mucus antibody values in this group did not rise above pretreatment levels. I. multifiliis infection induced a transient mucosal antibody response that coincided with the resolution of infection. Whether elicited by infection or intraperitoneal injection of i-antigen, the serum and mucus antibody responses of channel catfish immunized against I. multifiliis did not occur synchronously.

The SCAN domain is a highly conserved dimerization motif that is vertebrate-specific and found near the N-terminus of C(2)H(2) zinc finger proteins (SCAN-ZFP). Although the function of most SCAN-ZFPs is unknown, some have been implicated in the transcriptional regulation of growth factors, genes involved in lipid metabolism, as well as other genes involved in cell survival and differentiation. Here we utilize a bioinformatics approach to define the structures and gene locations of the 71 members of the human SCAN domain family, as well as to assess the conserved syntenic segments in the mouse genome and identify potential orthologs. The genes encoding SCAN domains are clustered, often in tandem arrays, in both the human and mouse genomes and are capable of generating isoforms that may affect the function of family members. Twenty-three members of the mouse SCAN family appear to be orthologous with human family members, and human-specific cluster expansions were observed. Remarkably, the SCAN domains in lower vertebrates are not associated with C(2)H(2) zinc finger genes, but are contained in large retrovirus-like polyproteins. Collectively, these studies define a large family of vertebrate-specific transcriptional regulators that may have rapidly expanded during recent evolution.

Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration. The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood. Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells. Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein. Under resting conditions, the protein is phosphorylated at S247 and S286. In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228. Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain. These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.

The SCAN domain is a conserved region of 84 residues found predominantly in zinc finger DNA-binding proteins in vertebrates. The SCAN domain appears to control the association of SCAN domain containing proteins into noncovalent complexes and may be the primary mechanism underlying partner choice in the oligomerization of these transcription factors. Here we have overexpressed, purified, and characterized the isolated SCAN domain (amino acids 37-132) from ZNF174. Both size exclusion chromatography and equilibrium sedimentation analysis demonstrate that the ZNF174 SCAN domain forms a homodimer. Circular dichroism shows that the isolated SCAN domain dimer has approximately 42% alpha-helix. Thermal denaturation experiments indicate that the SCAN domain undergoes a single reversible unfolding transition with a T(m) of over 70 degrees C. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration, consistent with a two-state unfolding transition in which folded dimer is in equilibrium with unfolded monomer. These findings demonstrate that the isolated SCAN domain forms a stable dimer and support a model in which the SCAN domain is capable of mediating the selective dimerization of a large family of vertebrate-specific, zinc finger-containing transcription factors.

For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another.

Milk was collected at 24 h intervals through 120 h after treatment from lactating dairy cows treated with antibiotics. Antibiotic residues were determined qualitatively by microbiological assays utilizing Bacillus stearothermophilus . Intrauterine infusion of antibiotics resulted in the lowest percentage of milk samples positive for residues. A high percentage of samples was positive for residues 24 and 48 h after intramuscular injection of antibiotics; however, most samples were negative by 72 h after treatment. Intramammary therapy resulted in a high proportion of samples positive for residues at 24 and 48 h after treatment, and some samples were positive 96 to 120 h after treatment. Samples from treated quarters were often positive when corresponding composite milk samples were negative. Treatment with more than one antibiotic by multiple routes resulted in the highest percentage of samples positive for residues for the longest time. Any variation of antibiotic dosage, duration of treatment, or use of multiple antibiotics should alert dairy producers of the probability of antibiotics being excreted in milk beyond recommended withdrawal times.

Bovine mammary glands were inoculated intracisternally with a streptomycin-resistant (SR) strain of Corynebacterium bovis to determine the number of colony-forming units (CFU) required to induce colonization and to maintain persistence of C bovis colonization throughout lactation and involution. Streptomycin resistance was used as a strain marker. Uninfected quarters in cows during midlactation were challenge exposed with successively higher numbers of SR C bovis until all quarters became colonized. Inoculum containing 790 CFU of SR C bovis established colonization in only 7 of 38 quarters. Colonization persisted in only 4 of these quarters by 23 days after inoculation. Eleven quarters were reinoculated with higher numbers of SR C bovis, and all became colonized by the time challenge-exposure inoculum contained 8 X 10(4) CFU. Colonization persisted throughout the 93-day experimental period. Somatic cell counts were significantly (P less than 0.01) higher in SR C bovis-colonized quarters after inoculation than before. Sixteen additional quarters were inoculated with a mean number of 8 X 10(4) CFU of SR C bovis 7 days before suppression of lactation. All quarters became colonized, and SR C bovis was shed during the experimental period; throughout the nonlactating and peripartum periods, high numbers of SR C bovis in pure culture were shed from 13 of 16 quarters.