Prevalence and Antimicrobial Resistance of Nontyphoidal Salmonella enterica from Head Meat and Trim for Ground Product at Pork Processing Facilities.

ABSTRACT: Pork head meat may harbor Salmonella and contaminate other carcass by-products during harvest and fabrication. A large pork processing plant in the United States was sampled bimonthly for 11 months to determine the concentration, prevalence, seasonality, serotype diversity, and antimicrobial susceptibility of Salmonella enterica isolated from cheek meat and head trim of swine carcasses. Each collection consisted of 25 samples on two consecutive days in the morning and afternoon shifts, for a total of 100 cheek meat and 100 head trim samples each month. Tissues were cultured for Salmonella by using restrictive media and enrichment techniques, and a subset of isolates was serotyped, analyzed for antimicrobial susceptibility, and genome sequenced. Salmonella postenrichment prevalence did not differ (P = 0.20) between cheek meat (63%) and head trim (66%). Postenrichment prevalence differed (P < 0.05) by month (January, 94%; March, 80%; May, 54%; July, 59%; September, 47%; and November, 55%) and by processing shift (morning, 68%; afternoon, 62%). The subset (n = 618) of isolates selected for serotyping yielded 21 distinct serotypes: Typhimurium (49%), Infantis (10%), Heidelberg (8%), I 4,[5],12:i:- (8%), and 17 other types (≤5%). In total, 407 multidrug-resistant (MDR; resistance to three or more antibiotic classes) isolates were identified. There were 120 isolates that exhibited the penta-resistant ACSSuT phenotype. In addition, 113 isolates exhibited decreased susceptibility to ciprofloxacin (MIC ≥ 0.12 µg/mL). Resistance genes blaCARB, blaSHV, blaTEM, aac(6')-Ib-cr,qnrB, sul2, and dfrA were expressed in numerous MDR Salmonella isolates. The data herein suggest that pork products from the head, compared with data reported for carcasses, may have a relatively high prevalence of Salmonella with diverse serotypes and MDR.

Evaluation of a Preharvest Bacteriophage Therapy for Control of Salmonella within Bovine Peripheral Lymph Nodes.

ABSTRACT: A series of proof-of-concept studies were developed to determine whether a commercial bacteriophage cocktail could be utilized for the mitigation of Salmonella in bovine peripheral lymph nodes (LNs). The first objective sought to determine whether exogenous phage could be isolated from the LNs following administration. If isolation were successful, the second objective was to determine whether the phage in the LNs could effectively reduce Salmonella. Salmonella Montevideo was inoculated intradermally at multiple sites and multiple times, followed by delivery of the phage cocktail subcutaneously in two injections around each of the right and left prescapular and subiliac LNs. At the conclusion of each study, animals were euthanized, and the popliteal, prescapular, and subiliac LNs were examined. The inoculated phage was successfully isolated from the LNs; transmission electron microscopy revealed phages in the LNs of the treated cattle, and these phages were identical to those in the cocktail. Levels of phage were higher (P < 0.01) in the prescapular and subiliac LNs in the phage-treated than in the control cattle. In subsequent studies, the protocols were modified to increase Salmonella and phage levels within the LNs. Compared with the first study, overall Salmonella levels were increased in the LNs, and phage treatment decreased (P < 0.01) Salmonella in the some of the LNs. Phage levels were numerically but not significantly increased (P = 0.12) in the treated cattle. The final study was modified, hypothesizing that a 48-h postmortem period before LN removal would facilitate phage-Salmonella interaction; however, no differences (P > 0.10) in Salmonella levels were found among treatments. Salmonella-specific phages administered to live cattle can translocate to the LNs; however, these phages had limited to no effect on Salmonella in these LNs under these experimental conditions.

Morphometrics of the Southern Green Stink Bug [Nezara viridula (L.) (Hemiptera: Pentatomidae)] Stylet Bundle.

The southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae), is a cosmopolitan pest of high-value cash crops, including cotton (Gossypium hirsutum L.; Malvales: Malvaceae). The pest can ingest and transmit disease-causing bacterial and fungal pathogens of cotton. We hypothesized that the size of the food canal may contribute to selective transmission, as observed in previous reports. The objective of this study was to examine food canal size and other morphometric parameters of the southern green stink bug stylet bundle at two locations (labium and head) to improve our understanding of factors that may contribute to pathogen transmission. For the food canal, females possessed significantly larger canals than males, major axes were significantly longer than minor axes, and canal sizes were numerically higher at the labium compared with the head. For salivary canal, mean axes lengths were similar between sexes and the head and labium. For both food and salivary canals, axes lengths were longer and area was larger at the labium compared with the head. These findings indicate the presence of a "funnel effect" with canals becoming narrower proximally. Sex and location significantly affected the size of the intact stylet bundle. Results indicate the food canal size was not a factor affecting previously observed selective passive transmission. Major and minor axes measurements, coupled with morphological observations of canal shapes and observed "funnel effect" in the food and salivary canals, improve our understanding of the hemipteran stylet bundle and its relationship with the insect's internal morphology.

Image contrast in sections of epoxy resin-embedded biological material: maintenance of a proper anhydride-epoxy ratio during tissue impregnation.

Epoxy resins are mixtures of several components formulated in exact proportions to assure proper polymerization. It is necessary that this composition be maintained throughout the tissue being infiltrated to avoid embedding artifacts. Unfortunately, the separate resin components will not penetrate the tissue at the same rate unless they all have the same viscosity and molecular size. Failure to meet these criteria will result in component separation during tissue infiltration and may result in severe embedding defects. This and several ancillary problems such as low tissue contrast, damage to diamond knives, and poor postsection staining are explored in this brief discussion.

Effects of chitin synthesis inhibitors on incorporation of nucleosides into DNA and RNA in a cell line from Manduca sexta (L).

Five putative chitin synthesis inhibitors (CSI) were tested to determine if they inhibited nucleoside incorporation into acid precipitable material in a cell line from Manduca sexta (L.). The results varied. Diflubenzuron (DFB) (100 micron) inhibited cytidine incorporation by 38%; EL-494 (100 micron) inhibited adenosine incorporation by 43%; Bay Sir 8514 (100 micron) inhibited uridine incorporation by 24%. Superdiflubenzuron (100 micron) was the worst inhibitor overall (18-22%) for the benzoylphenyl urea CSI. The triazine CSI, CGA 19255, was the best inhibitor tested with 60% inhibition for cytidine and 49% for adenosine incorporation into DNA and RNA. Examination of cells incubated with diflubenzuron by scanning electron microscopy revealed distinct external morphological changes. Transmission electron microscopy showed that crystalline structures accumulated in the cytoplasm of cells treated with DFB. The crystalline structures were assumed to be diflubenzuron and they persisted even after diflubenzuron was removed from the medium.

Differential expression of adhesion molecules by chicken heterophils activated in vivo with Salmonella enteritidis-immune lymphokines.

Chicken heterophils activated in vivo following the intraperitoneal (i.p.) administration of Salmonella enteritidis-immune T lymphokines (SE-ILK) have been implicated in the protection against SE organ invasion. SE-ILK induces a heterophilia and directly (or indirectly) activates the granulocytes. The invasion of SE provides the secondary signal for directing activated heterophils to the site of bacterial invasion. We examined the mechanism of adherence within the avian heterophil system using an in vitro bovine serum albumin (BSA) matrix in which neutrophil adherence is primarily CD11/CD18 integrin mediated in mammalian systems. Activated heterophils displayed a four-fold increase in receptor-mediated adherence in vitro to BSA-coated slides as compared to control heterophils from PBS-injected birds. The increased adherence of activated heterophils can be partially blocked by either anti-alpha M (CD11b) or anti-beta 2 (CD18) antibodies in a dose dependent manner. Anti-alpha 3 (CD49c) antibody partially blocked adherence of both normal and activated cells. Fluorescence-activated cell scanning (FACS) analysis of the heterophils shows that both control and SE-ILK-activated heterophils collected at 4 h post injection with SE-ILK or PBS display similar amounts of integrin alpha 3 on their surface. This integrin is constitutively expressed and is responsible for the in vitro adherence of both groups. However, antibodies to the Mac-1 complex (CD11b/CD18) block only the adherence of SE-ILK-stimulated heterophils. Thus, the CD11b/CD18 heterodimer is apparently up regulated in response to the injected SE-ILK and plays a major role in the adherence of activated heterophils. Our studies in chickens parallel human and mouse studies showing the importance of the beta 2 integrins in adherence of activated cells.

Prevalence of antimicrobial resistance in Salmonellae isolated from market-age swine.

Antimicrobial resistance levels were examined for 365 Salmonella isolates recovered from the lymph nodes (n = 224) and cecal contents (n = 141) of market-age swine at slaughter. Antimicrobial resistance testing was performed by disk diffusion using 13 antibiotics common in the treatment of disease in human and veterinary medicine. Although none of the antibiotics tested were used subtherapeutically within the last 5 years on the farms sampled, resistance to chlortetracycline, penicillin G, streptomycin, and sulfisoxazole was common. Penicillin G resistance was significantly more frequent (P = 0.03) and sulfisoxazole resistance was significantly less frequent (P < 0.01) in lymph node versus cecal isolates. Multidrug resistance was observed among 94.7% of the lymph node isolates and 93.5% of the cecal isolates. The most frequent multidrug resistance pattern included three antibiotics-penicillin G, streptomycin, and chlortetracycline. Isolates in somatic serogroup B, and more specifically, Salmonella Agona and Salmonella Schwarzengrund isolates, were often resistant to a greater number of antibiotics than were isolates in the other serogroups. Streptomycin, sulfisoxazole, ampicillin (lymph node isolates), and nitrofurantoin (cecal isolates) resistance levels differed significantly between somatic serogroups. The prevalence of penicillin G-, streptomycin-, and sulfisoxazole-resistant isolates differed significantly between serovars for both lymph node and cecal isolates. Results of this study suggest that a correlation exists between the somatic serogroup or serovar of a Salmonella isolate and its antimicrobial resistance status, which is specific to the antibiotic of interest and the source of the isolate (lymph node versus cecal contents).

Diminution of Campylobacter colonization in neonatal pigs reared off-sow.

Pigs may be a natural reservoir of Campylobacter and can be colonized as early as 24 h after birth. The purpose of the present study was to evaluate what effect early removal of piglets from Campylobacter-positive sows has on Campylobacter prevalence in neonates. In two trials, piglets were removed from sows within 24 h of birth and were reared in nurseries isolated from sows for 21 days. From the neonates rectal swabs were cultured for Campylobacter, and Campylobacter status of the isolated piglets was compared to that of littermates reared on sows. The nurseries consisted of wire-floored farrowing crates that were equipped with heaters and self-feeders. In trial I, the Campylobacter prevalence in nursery-reared piglets was 13 of 14 on day 2 and 0 of 14 on day 20. Campylobacter prevalence in the sow-reared piglets was 8 of 9 from days 2 to 20. In trial II, 12 of 29 on day 2, and 5 of 26 on day 20, of the nursery-reared piglets were culture positive for Campylobacter. For the sow-reared piglets, Campylobacter prevalence was 7 of 15 on day 1 and 15 of 15 (100%) on day 20. These data suggest that successful permanent colonization of the gut by Campylobacter is probably related to constant exposure of piglets to Campylobacter-positive feces. Campylobacter prevalence may be diminished in neonates that are reared off-sow in isolated nurseries.

Genotypic variation among arcobacter isolates from a farrow-to-finish swine facility.

Arcobacter spp. were isolated from nursing sows and developing pigs on three farms of a farrow-to-finish swine operation and market-age pigs at slaughter. Isolates were identified by polymerase chain reaction and genotypic fragment patterns were examined by pulsed-field gel electrophoresis (PFGE). Incidences of Arcobacter-positive samples increased progressively as the pigs aged, resulting in all of the pens at the end of the growth cycle in the finishing barn containing Arcobacter-positive feces. However, only 10 of 350 cecal samples from slaughtered pigs were positive. There was little similarity between genotypic patterns for Arcobacter collected from the three farms. The level of genotypic variation revealed by PFGE suggested that pigs in this farrow-to-finish operation were colonized by multiple Arcobacter parent genotypes that may have undergone genomic rearrangement, common to members of Campylobacteraceae, during successive passages through the animals. Additionally, the level of genotypic diversity seen among Arcobacter isolates from farms of a single farrow-to-finish swine operation suggests an important role for genotypic phenotyping as a source identification and monitoring tool during outbreaks.

Effects of feed withdrawal and transport on cecal environment and Campylobacter concentrations in a swine surgical model.

The objective of the present study was to evaluate how feed withdrawal and transportation influenced the cecal environment and cecal populations of Campylobacter in swine. Four miniature Yucatan gilts (8.8 kg), naturally infected with Campylobacter jejuni, were surgically implanted with cecal cannulas. The gilts were fasted for 48 h. Samples of cecal contents were collected for 7 days prior to and for 7 days after the fast, and mean values were determined for pH, volatile fatty acids (VFA), and CFU enumeration of C. jejuni. This was replicated three times. In another trial, gilts (full-fed) were transported in a livestock trailer for 4 h and cecal samples were collected before and after transport and analyzed for pH, VFA, and CFU. Following a 48-h fast, cecal pH increased (P < 0.05) by 1 unit; acetic and propionic acids decreased (P < 0.05) by 61% and 71%, respectively; and there was a twofold log10 increase (P < 0.05) in CFU/g cecal content of C. jejuni. Values of pH, VFA, and CFU of C. jejuni did not change in cecal samples from gilts following transportation. These data are important for food safety considerations because feed withdrawal, commonly associated with shipping and slaughter, can increase Campylobacter concentrations in the pig intestinal tract.

Comparison of GN Hajna and tetrathionate as initial enrichment for salmonellae recovery from swine lymph nodes and cecal contents collected at slaughter.

An epidemiologic survey was conducted to determine the prevalence of salmonellae in swine from 5 farms of an integrated swine operation. The purpose of this study was to evaluate the recovery efficiencies for salmonellae from swine lymph nodes and cecal contents when GN Hajna and tetrathionate were compared as initial enrichments. Salmonellae were isolated from 61% of 645 pigs at slaughter; 324 positive cultures were from lymph nodes, and 224 were from cecal contents. Frequently, pigs had salmonellae isolated from both the lymph nodes and cecal contents. Total isolations, regardless of source, were similar for GN Hajna (247) and tetrathionate (301). There was no difference (P > 0.05) in the number of isolations from lymph nodes when GN Hajna enrichment was compared with tetrathionate enrichment (174 vs. 150). However, there was a significant (P < 0.05) advantage of utilizing tetrathionate when compared with GN Hajna for isolations from cecal contents (151 vs. 73).

In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells.

Primary cultures of Anaplasma marginale infected erythrocytes were used to determine conditions for in vitro cultivation of the rickettsia. The infected erythrocytes that were maintained by regular addition of Glasgow's MEM with fetal calf serum and uninfected erythrocytes showed a 1-5% increase in percent infected erythrocytes on the evaluation of Giemsa stained smears. This increase in parasitemia resulted in up to 70% change in the number of infected erythrocytes. Co-culture of the infected erythrocytes with endothelial cell monolayers allowed for longer maintenance with the parasitemia ranging from 5-13% through four passages over 16 weeks. Examination of cultures using transmission electron microscopy showed initial bodies within the erythrocytes at 10 days after the initial passage of the primary culture. The endothelial cell monolayers in the co-cultures contained multiple initial bodies. We have demonstrated that A. marginale can be grown for a limited number of passages in the co-culture system, which will facilitate the development of a continuous culture of the organism.

Ultrastructure of Hepatozoon canis in the dog.

The ultrastructure of several stages of Hepatozoon canis found in dogs with clinically diagnosed infections was determined using transmission electron microscopy. Stages were found in skeletal muscle tissue that corresponded to the 'onion skin' cyst stage, as described at the light microscopic level, and were composed of an electron-transparent material that appeared to radiate from a central core. Larger cysts, walled off by fibroblasts, contained a transformed host cell located centrally within them. The parasitic stage within these transformed cells contained numerous organelles including mitochondria and Golgi apparatus, and was singularly nucleated. In cardiac muscle, a meront was observed which produced merozoites by ectomerogony. Adjacent to this meront was a granuloma containing merozoites within mononuclear phagocytes which may either serve as a reservoir of parasites for reinfection of the host or differentiate into the circulating gamont stage of the parasite. Gamonts were found within parasitophorous vacuoles inside circulating neutrophils. They had a condensed cytoplasmic appearance and were extremely electron dense with respect to other observed parasite stages. In vitro cultivation of parasitized neutrophils resulted in the appearance of a stage of the parasite with altered ultrastructure compared with gamonts found in circulating neutrophils. This stage was judged to be a possible gamete stage of the parasite.

Effect of mannose on Salmonella typhimurium-mediated loss of mucosal epithelial integrity in cultured chick intestinal segments.

The effect of incubating Salmonella typhimurium and S. typhimurium cell-free extracts with isolated intestinal segments from 1-day-old chicks was evaluated using scanning electron microscopy. Incubation of segments with intact bacteria or with cell-free extract resulted in the loss of mucosal epithelial integrity after as little as 30 min incubation. Loss of mucosal epithelial integrity was evidenced by the complete shedding of the epithelium. The addition of 2.5% D-mannose to the incubation medium inhibited the loss of epithelial cells, whether intestinal segments were incubated with intact bacteria or with cell-free extract. These results indicate that S. typhimurium exerts a D-mannose-sensitive cytotoxic effect on the mucosal epithelium of isolated intestinal segments and that the cytotoxic effector is present in cell-free extracts of the bacteria.

Ultrastructural characteristics of Babesia odocoilei in vitro.

Babesia odocoilei continuously cultured in white-tailed deer (Odocoileus virginianus) erythrocytes was examined by transmission and scanning electron microscopy. Merozoites, trophozoites, intermediate-stage forms, and dividing forms were observed. Merozoites possessed a single nucleus, inner membrane complex, rhoptries, free ribosomes, rough endoplasmic reticulum, and single membrane-bound vesicles. Trophozoites lacked an inner membrane complex and rhoptries. Intermediate stages were characterized by distinct segments of inner membrane complex. Dividing forms ranged from cells with an elongated nucleus to mature daughter cells joined by a ringlike structure. Babesia odocoilei was characterized by its close proximity to the erythrocyte membrane, membranous structures resembling feeding organelles, and reproduction via a method resembling budding sensu stricto.

In vitro growth of Babesia bovis in white-tailed deer (Odocoileus virginianus) erythrocytes.

Babesia bovis cultured in bovine erythrocytes was passaged into white-tailed deer (Odocoileus virginianus) erythrocytes and medium containing either white-tailed deer serum or bovine serum. Deer erythrocytes supported the growth of the parasite only in the presence of bovine serum. Cryopreserved cultures were recovered successfully in white-tailed deer erythrocytes. By light and electron microscopy, B. bovis structure appeared similar in host cells of either species.

Ultrastructure of Babesia bovis sexual stages as observed in Boophilus microplus cell cultures.

Propagation of Babesia bovis in a Boophilus microplus cell line resulted in the appearance of the sexual stage of the parasite normally found only within tick intestine. These sexual stages, which possessed spike-like projections containing microtubules, were present in the medium and within cultured cells. Other ultrastructural characteristics of this sexual stage are described.

Carrier erythrocytes from white-tailed deer: morphology, osmotic fragility and survival of circulating sickled erythrocytes.

Carrier erythrocytes are used to disseminate drugs in the circulatory system of animals. Carrier erythrocytes prepared from white-tailed deer (Odocoileus virginianus) do not circulate well in vivo. Although carrier cells were prepared from sickle and non-sickle cells with no apparent differences, their 24-hour survival was only 10 per cent. Osmotic fragility of carrier cells was increased over that of normal deer erythrocytes. Unlike erythrocytes from other ruminants, deer carrier erythrocytes are extremely fragile. Scanning electron micrographs of deer erythrocytes (sickle or non-sickle) in different stages of carrier cell preparation revealed no morphological differences. These data suggest that carrier cells from deer would not be amenable for use in long-term dissemination of drugs.

Properties of microcytic caprine carrier red blood cells: their preparation and in vivo survival.

Dialysis encapsulation was used to prepare caprine-carrier RBC. Optimal conditions for encapsulation are described in which 23% of added [14C]sucrose was entrapped. Carrier RBC were stable in vitro releasing only 5% of entrapped substance in 24 hour. Injected-carrier RBC circulated with a 47-day half-life. Although markedly smaller in size (10 fl vs 15 fl), caprine-carrier RBC were morphologically identical to normal RBC.

Ultrastructure of hepatic and renal lesions in chickens fed aflatoxin.

Male broiler chicks were given feed and water ad libitum from hatching through 3 weeks of age. The feed contained 0, 1.25, 2.5, and 5.0 micrograms of aflatoxin/g of feed. The chicks were killed by cervical dislocation and specimens of liver and kidney were obtained for electron microscopy on days 3, 6, 9, 17, and 21. In chicks fed 5.0 micrograms of aflatoxin, the primary lesions in liver were hepatocellular lipidosis, enlargement of bile canaliculi, reduction in mitochondrial size, mild lymphocytic infiltration, and hepatocellular degeneration and necrosis. Similar lesions were noticed in some chicks fed 2.5 micrograms of aflatoxin, but none was observed in chicks fed at 1.25 micrograms of aflatoxin. At 5 micrograms of aflatoxin, the most consistent lesion in the kidney was thickening of the glomerular basement membrane. Similar glomerular lesions were observed at 2.5 micrograms of aflatoxin, but not at 1.25 micrograms of aflatoxin. Some foot processes of the glomerular epithelial cells were poorly developed. Fusion of foot processes was not observed and fibrous material was not evident in the basement membrane. The pseudopodia of endothelial cells lining the thickened basement membrane were depleted in number or were absent. Degenerative changes also were observed in the cells of the proximal convoluted tubules, but these were less consistent than those of the glomerulus.