Diaphragm Muscle Samples Research Articles

Meat Juice and Oral Fluid as Alternatives to Serum for Aujeszky Disease Monitoring in Pigs.

Aujeszky Disease Virus (ADV) is a double-stranded DNA virus with a lipoprotein envelope. The natural hosts of the infection are Suidae, but the virus can infect many other mammals. The gold-standard method identified by the WOAH for the diagnosis of Aujeszky disease is the ELISA method. The objective of this study was to compare the performance of meat juice and oral fluid matrices using a commercial ELISA kit designed for serum. A total of 80 blood and oral fluid samples were collected from four pig farms selected for this study. Diaphragm muscle samples of about 100 g and blood samples were collected from 213 animals at the abattoir. These biological matrices were collected from the same animals and tested using a competitive ELISA kit to detect antibodies against ADV. The relative accuracy of the meat juice compared to that of the serum was 96.7% (95% CI: 93.3-98.7%), with 206 correct results out of 213. The relative accuracy of the oral fluid compared to that of the serum was 61.3% (95% CI: 49.7-71.9%), with 58 correct results out of 80. Meat juice has a better combination of sensitivity and specificity than oral fluid. The usage of meat juice in routine diagnostic examinations could be achieved after further investigations to optimize the procedure.

Molecular identification of Sarcocystis species in sika deer (Cervus nippon) of free-ranging populations in Germany and Austria.

In this study, for the first time, Sarcocystis species were identified molecularly in sika deer (Cervus nippon) that form free-ranging populations in several countries of Europe. Diaphragm muscle samples from 151 deer from 10 populations in Germany and Austria were examined for sarcocysts. By one-gram methylene-blue staining, sarcocysts were recorded in samples of 114 animals (75.5%) which originated from all populations. Sarcocysts were more often (p < 0.0001) recorded in yearling and adult deer than in calves. Infection intensity was generally low with ~ 70% of the sarcocyst positive deer harbouring ≤ 10 sarcocysts per 1-gram diaphragm muscle. Based on cox1 sequence comparison, 10 species of Sarcocystis, all previously reported parasitizing cervids, were identified: S. elongata, S. entzerothi, S. hjorti, S. iberica, S. japonica, S. linearis, S. morae, S. pilosa, S. silva and S. truncata. The prevailing S. hjorti was detected in sika deer of all 10 populations. The identification in sika deer of S. hjorti, S. iberica, S. elongata, S. linearis, S. morae and S. silva constitutes new host records. With the additional species records of this study, the highest number of Sarcocystis species, at least 16, was identified in this host.

Aging Selectively Reduces Mitochondrial Volume Density and Respiratory Capacity in Type IIx/IIb Diaphragm Muscle Fibers

Diaphragm muscle (DIAm) sarcopenia involves selective atrophy of type IIx/IIb DIAm fibers. In addition, there is an age‐related decrease in the maximum velocity of the succinate dehydrogenase reaction (SDHmax) in type IIx/IIb fibers. SDH is a key enzyme of the tricarboxylic acid (TCA) cycle as well as complex II of the electron transport chain (ETC). These results suggest that sarcopenia may reduce mitochondrial volume density in type IIx/IIb DIAm fibers and thereby reduce SDHmax. Alternatively, sarcopenia may reduce the intrinsic respiratory capacity of mitochondria in type IIx/IIb DIAm fibers. We hypothesized that there is an age‐related selective reduction in mitochondrial volume density and SDHmax per mitochondrial volume in type IIx/IIb DIAm fibers. In 24 Fischer 344 rats (6‐mo: n=6 female, n=6 male; 24‐mo: n=6 female, n=6 male), DIAm samples were dissected from the mid‐costal region and rapidly frozen at optimal sarcomere length. In 10 mm cross‐sections, DIAm fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Mitochondria were labelled using Mitotracker Green and imaged using 3D confocal microscopy (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. Each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI=SA3/16µ2V2) using ImageJ and NIS elements. In alternate 6 mm sections, SDHmax was measured using a quantitative histochemical assay in which accumulation of nitro blue tetrazolium diformazan (NBTdfz) was measured in DIAm fibers every 15 s for 10 min. Consistent with previous findings, type IIx/IIb DIAm fibers were ~36% smaller in 24‐mo compared to 6‐mo rats (P&lt;0.0001), whereas there were no age‐related differences in cross‐sectional areas (CSA) of type I and IIa fibers. Mitochondrial volume density was lower in IIx/IIb fibers of 24‐mo compared to 6‐mo rats (P&lt;0.0001), whereas there were no age‐related differences in type I and IIa fibers. The MCI in type IIx/IIb fibers was lower (greater fragmentation) in 24‐mo compared to 6‐mo rats (P&lt;0.0001), whereas the MCIs of type I and IIa fibers were similar across ages. Finally, SDHmax per fiber volume was lower in type IIx/IIb DIAm fibers in 24‐mo compared with 6‐mo rats (P&lt;0.0001), whereas there were no age‐related differences in type I and IIa fibers. When SDHmax was normalized for mitochondrial volume, there was no age‐related difference in the intrinsic mitochondrial respiratory capacity in type IIx/IIb fibers. Therefore, aging results in reduced mitochondrial volume density and increased fragmentation in type IIx/IIb DIAm fibers, but no change in the intrinsic respiratory capacity of mitochondria.

Molecular detection and genetic characterization of Toxoplasma gondii in pork from Chongqing, southwest China

Raw or undercooked meat is an important source of Toxoplasma gondii infection in China, but there is little research data on these infections in Chongqing. This study determined the prevalence of T. gondii, and its genotypes, in pork. A total of 1,223 diaphragm muscle samples were collected from eight slaughterhouses and 79 markets and detected by PCR amplification of the ITS gene. All of the positive samples were used for genotype identification by PCR-RFLP with 11 genetic markers. The total positive rate of T. gondii in Chongqing pork was 8.7%, and differences in T. gondii infection rates were found between different districts (0%–23.3%), seasons (e.g., 4.3% from Spring, 7.3% from Summer, 11.4% from Autumn, 12.0% from Winter) and years (2.7%–14.3%). Six samples were successfully genotyped, of which one was identified as ToxoDB#9 and five were ToxoDB#9-like. This was the first continuous study about the prevalence of T. gondii in pork in Chongqing for several years. Slaughterhouses in different districts, pork source, farm scale, season and year were potential risk factors for T. gondii contamination by the univariate logistic regression, and using multivariate logistic regression districts, pork source and year were the independent risk factor. These data may help reducing the levels of toxoplasmosis in pigs and humans in Chongqing.

Seroprevalence of Toxoplasma gondii and Salmonella in Hunted Wild Boars from Two Different Regions in Switzerland.

Simple SummaryWild boars are widely distributed in the northern and southern wooded regions of Switzerland and are popular for their meat. Wild boars can carry a variety of parasites, bacteria, and viruses that could infect humans and domestic animals. In this study, we focused on two important pathogens, the parasite Toxoplasma gondii and the bacterium Salmonella. We used enzyme-linked immunosorbent assay (ELISA) tests to detect antibodies to these pathogens in diaphragm tissue samples from hunted wild boars from two different regions of Switzerland. While the seroprevalence of T. gondii antibodies was similar for animals from the northern and southern region (29% and 37%, respectively), Salmonella seropositivity was very much higher in wild boars from the northern area (52%) than among animals from the south (5%). This may be related to the wild boar density, which may in turn be a risk factor for domestic animals and humans living in the same area. Pathogens in wild boars are of public health significance as a potential source of meat-borne diseases in humans.Toxoplasma gondii and Salmonella are zoonotic foodborne pathogens that may be transmitted to humans through the consumption of raw or undercooked meat, including game. The aim of this study was to determine the seroprevalence of T. gondii and Salmonella antibodies in wild boars in two different regions in Switzerland. During the hunting season of 2020, a total of 126 diaphragm muscle samples of hunted wild boars were collected and the meat juice of these samples was analysed for pathogen-specific IgG antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. The overall seroprevalences were 35% for T. gondii and 17% for Salmonella, respectively. In general, seropositivity increased with the age of the animals. Seroprevalences of T. gondii were similar for animals from the northern region (29%) to those from the southern region (36.8%), indicating that T. gondii is widespread in the sylvestrian environment. By contrast, Salmonella seropositivity was remarkably higher in wild boars from the north (52%) compared with those from the south (5.3%). The high occurrence of Salmonella may represent a risk of transmission to compatriot domestic animals such free-range farmed pigs as well as to humans. Further, meat of hunted wild boars may present a source of human toxoplasmosis or salmonellosis.

Molecular identification of Sarcocystis species in diaphragm muscle tissue of European mouflon (Ovis gmelini musimon) from Austria.

Previous morphological studies suggested that mouflon may have sarcocysts similar to those of sheep. However, to date, no molecular-based studies of the species of Sarcocystis infecting mouflon have been done. The present study identified Sarcocystis species in diaphragm muscle samples from 20 European mouflon (Ovis gmelini musimon). Molecular identification using the cox1 sequence analysis was performed on sarcocysts excised from muscle tissue and on DNA from digested muscle samples. Both frequency and intensity of infection in mouflon were high with 19 of 20 animals testing Sarcocystis positive and > 50 cysts per gram of tissue recovered from 10 of the 19 Sarcocystis positive animals. Molecular analysis revealed dominant Sarcocystis tenella (18/19 animals) and Sarcocystis arieticanis (1/19 animals), whose known intermediate hosts are sheep. In addition, Sarcocystis capracanis, which is known to form sarcocysts in goats, was detected in two animals. The results of this study demonstrated the digestion method to be superior over the direct isolation of sarcocysts for the molecular identification of Sarcocystis species in a certain host. Future research of Sarcocystis diversity in wild ovine and caprine species is needed.

Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

BackgroundCattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis.MethodsTo reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification.ResultsSarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses.ConclusionsOur study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis.Graphical

Epidemiology of Leishmania Carriers in Tan Chang County, Gansu Province, China.

BackgroundLeishmaniasis is a regional infectious disease caused by the bite of Leishmania-carrying sandflies. The clinical symptoms include prolonged fever, spleen enlargement, anemia, emaciation, leukopenia, and increased serum globulin levels. If not appropriately treated, patients may die of complications caused by leishmaniasis within 1–2 years after the onset of the illness. Therefore, further investigation of the mechanisms of infection by this pathogen is required. Here, an epidemiological study of Leishmania carriers was conducted. The potential mechanism of infection through domestic animals as carriers of the parasite was investigated to identify potential reservoir hosts for Leishmania. MethodsThe rK-39 strip test was performed on blood samples from previously infected patients. Blood samples were collected from the patients and their families. The blood, liver, spleen, and diaphragm muscle samples were collected from livestock. To perform nested polymerase chain reaction (PCR), DNA was extracted and the internal transcribed spacer sequence was used. The amplified products were then subjected to restriction fragment length polymorphism and phylogenetic analyses.ResultsAmong previously infected patients, 40% (12/30) showed positive results in the rK-39 strip test. The nested PCR positive rates for previously infected patients/relatives and livestock samples were 86% (77/90) and 80% (8/10), respectively. Moreover, the phylogenetic analysis showed that the pathogen was Leishmania infantum. Dogs, patients, and domesticated animals carrying Leishmania were found to be a potential source of infection for leishmaniasis.ConclusionsThe results of this study provide a basis for developing disease prevention and control strategies for leishmaniasis.

Molecular identification of two Sarcocystis species in fallow deer (Dama dama) from Lithuania

Due to the lack of molecular research conducted, little is known about Sarcocystis species diversity in the fallow deer (Dama dama). Until now, Sarcocystis jorrini and Sarcocystis morae were described to form sarcocysts in the muscles of this host. In the present study diaphragm muscle samples of free-ranging fallow deer from Lithuania were investigated for Sarcocystis species. Sarcocysts were detected in 39 out of 48 (81.3%) fallow deer examined. Under a light microscope two types of sarcocysts having hair-like and finger-like protrusions were observed. Based on DNA sequence analysis of cox1 and 18S rDNA, two species, S. morae and Sarcocystis entzerothi were identified. In prior studies, the latter species was only detected in Lithuanian roe deer (Capreolus capreolus) and in sika deer (Cervus nippon). The haplotype network of S. morae sequences specified close relationships between haplotypes found in the same country. According to current knowledge, the fallow deer is characterised by low Sarcocystis species richness as compared with other cervid species from Europe.

Occurrence of selected zoonotic food-borne parasites and first molecular identification of Alaria alata in wild boars (Sus scrofa) in Italy.

Wild boar is a source of human infections with zoonotic pathogens, including food-borne parasites. With the aim of a characterization of the human exposure risk, a survey on wild boars intended for human consumption was planned, selecting three pathogens, Toxoplasma gondii, Alaria alata, and Trichinella spp., as markers of meat infection. Diaphragm muscle samples from 100 wild boars hunted in Piedmont region (Northern Italy) in two hunting seasons (2015-2016) were collected. Concerning T. gondii, a combined approach of antibody detection and molecular techniques with genotyping was performed. For the detection of A. alata and Trichinella spp., the larva migration technique and the magnetic stirrer method were employed, respectively; in addition, molecular confirmation of the morphological identification of the recovered specimen was performed. Anti-T. gondii antibodies were found in meat juice samples (43.3%) and T. gondii DNA (type II) was detected in three animals (7.1%) out of 42 seropositive examined. In none of the sampled wild boars (0%), Trichinella spp. larvae were found, whereas one animal (1%) scored positive to A. alata mesocercariae. The molecular diagnosis proved the morphological identification of the trematode. This is the first finding of A. alata in Italian wild boar population. The present study confirmed the role of wild boars as a source of parasitic zoonotic diseases and thus the risk derived for humans posed by the consumption of game meat. Considering the zoonotic implications, the results underline the importance of monitoring and surveillance of zoonotic parasites in Italian wild boar populations.

Detection, seroprevalence and antimicrobial resistance of Yersinia enterocolitica and Yersinia pseudotuberculosis in pig tonsils in Northern Italy

Yersiniosis is the third most common reported zoonoses in Europe, with Y. enterocolitica and Y. pseudotuberculosis responsible for 98.66% and 0.94% of the confirmed human cases in 2013. From June 2013 to October 2014, 201 pigs at slaughter belonging to 67 batches were tested for Y. enterocolitica and Y. pseudotuberculosis in tonsils. Diaphragm muscle samples were tested for antibodies against Yersinia by a commercially available ELISA test. Y. enterocolitica 4/O:3 was detected in 55/201 pig tonsils (27.4%; 95% CI 23.1–37.1). The positive pigs came from 38/67 batches (56.7%) and were reared in 36/61 farms (59.0%). There was no statistical difference between farrow-to-finish and finishing farms. The mean count of Y. enterocolitica was 3.56±0.85log10CFU/g with a minimum of 2.0log10CFU/g and a maximum of 4.78log10CFU/g. Y. pseudotuberculosis was isolated from 4/201 pig tonsils (2.0%; 95% CI 0.0–4.5). Three isolates belonged to serotype O:3 and one to serotype O:1. The positive pigs belonged to 4/67 batches (6.0%) and came from finishing farms only. Y. pseudotuberculosis could be enumerated in one sample only (4.27log10CFU/g). The ELISA test demonstrated that 56.1% of the meat juice samples were positive for Yersinia antibodies. Serological positivity was found in 67.9% (36/53) of the Y. enterocolitica- and 75.0% (3/4) of the Y. pseudotuberculosis positive pigs. A significant association was found between serological results and the presence of Y. enterocolitica in tonsils (OR=1.97, p=0.044).All the Y. enterocolitica 4/O:3 isolates were susceptible to amoxicillin-clavulanic acid, gentamicin, ceftazidime, ertapenem and meropenem, 94.5% to cefotaxime, 89.1% to kanamycin and 78.2% to tetracycline. The highest resistance rates were observed for ampicillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Y. pseudotuberculosis strains were sensitive to all the antimicrobials tested, i.e. amoxicillin, amoxicillin/clavulanic acid, azithromycin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulphonamide, tetracycline and ticarcillin.The study shows that Italian fattening pigs are frequently infected with human pathogenic Y. enterocolitica 4/O:3. Although the isolation rate is slightly lower than in other European countries, the serological test demonstrates that the infection is widespread among pig population. In fact, seroprevalence is similar to other EU countries. The detection of Y. pseudotuberculosis serotypes O:1 and O:3 in pig tonsils is of concern.Since tonsils may represent a contamination source for pig meat at slaughter, further studies regarding human infections by both microbial species are strongly recommended.

Patterns and risks of trichinella infection in humans and pigs in northern Laos.

Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory–secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI) = 17.1–21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.

Determinación de residuos de oxitetraciclina en muestras de carne bovina

The aim of this study was to determine the presence of oxytetracycline (OTC) residues in 149 samples of diaphragm muscle of beef cattle, in a commercial slaughterhouse in Antioquia (Colombia). The samples were processed and purified through solid phase extraction. OTC detection was performed using high performance liquid chromatography (HPLC), the Maximum Residue Limits (MRL) of the European Union was used as reference (MRL > 100 ppb). Forty-nine percent of the samples (n = 73) presented OTC residues, 8% (n = 12) of samples contained amounts of OTC beyond the MRL. Age, breed and origin were not associated with the presence of oxytetracycline residues (p > 0.05). Prevalence of residues is higher among female than male (p = 0.06). It is concluded that although the prevalence of OTC residues in bovine meat evaluated was low, it is clear that the implementation of good farming practices, a system of epidemiological surveillance and training programs for professionals, livestock producers, and staff responsible for the animals should be strengthened, in order to guarantee safe food to consumers.

Effect of Intermittent Phrenic Nerve Stimulation During Cardiothoracic Surgery on Mitochondrial Respiration in the Human Diaphragm*

Recent studies have shown that brief periods of mechanical ventilation in animals and humans can lead to ventilator-induced diaphragmatic dysfunction, which includes muscle atrophy, reduced force development, and impaired mitochondrial function. Studies in animal models have shown that short periods of increased diaphragm activity during mechanical ventilation support can attenuate ventilator-induced diaphragmatic dysfunction but corresponding human data are lacking. The purpose of this study was to examine the effect of intermittent diaphragm contractions during cardiothoracic surgery, including controlled mechanical ventilation, on mitochondrial respiration in the human diaphragm. Within subjects repeated measures study. Operating room in an academic health center. Five subjects undergoing elective cardiothoracic surgery. In patients (age 65.6 ± 6.3 yr) undergoing cardiothoracic surgery, one phrenic nerve was stimulated hourly (30 pulses/min, 1.5 msec duration, 17.0 ± 4.4 mA) during the surgery. Subjects received 3.4 ± 0.6 stimulation bouts during surgery. Thirty minutes following the last stimulation bout, samples of diaphragm muscle were obtained from the anterolateral costal regions of the stimulated and inactive hemidiaphragms. Mitochondrial respiration was measured in permeabilized muscle fibers with high-resolution respirometry. State III mitochondrial respiration rates (pmol O2/s/mg wet weight) were 15.05 ± 3.92 and 11.42 ± 2.66 for the stimulated and unstimulated samples, respectively (p < 0.05). State IV mitochondrial respiration rates were 3.59 ± 1.25 and 2.11 ± 0.97 in the stimulated samples and controls samples, respectively (p < 0.05). These are the first data examining the effect of intermittent contractions on mitochondrial respiration rates in the human diaphragm following surgery/mechanical ventilation. Our results indicate that very brief periods (duty cycle ~1.7%) of activity can improve mitochondrial function in the human diaphragm following surgery/mechanical ventilation.

Survey of Trichinella infections in domestic pigs from northern and eastern Henan, China

The aim of this work was to investigate the current situation of Trichinella infections in swine in the cities of Anyang and Shanqiu in the Henan province historically designated as trichinellosis-free. A total of 475 diaphragm muscle samples were collected from 2010 to 2011 and examined by trichinelloscopy and artificial digestion. No Trichinella larvae were detected by trichinelloscopy; however, using the digestion method, 3.79% (18/475) of domestic pigs were deemed positive for Trichinella. Among the 475 pigs examined, 112 from an industrialized pig farm were negative. However. Trichinella larvae were detected in 10% (9/90) of pigs from small pig farms, which was significantly higher than the 3.3% (9/273) of pigs found positive from backyard farms (P<0.05). The larval burdens in infected animals ranged from 0.1 to 1.58 larvae per gram. The larvae were identified by multiplex PCR as Trichinella spiralis. Our study confirms the existence of porcine trichinellosis in northern and eastern parts of Henan. The results will be useful for evaluating the risk of infection for humans. Given this new found data, public health officials should consider implementing strategies to eliminate human transmission.

Effect of nitrofen in the final stages of development of the diaphragm muscle in rats

To evaluate the expression of myosin in muscle fibers of the diaphragm in experimental congenital diaphragmatic hernia (CDH). Fetuses of pregnant rats were divided into four groups: External Control (EC), composed of non-manipulated rats; Nitrofen, composed of pregnant rats that received 100 mg of nitrofen (2,4-dichloro-4'nitrodiphenyl ether) diluted in olive oil on gestational day (GD) 9.5, whose fetuses developed CDH (N+) or not (N-), and Olive Oil Placebo (OO), composed of pregnant rats that received the oil on the same GD. The fetuses were collected on GD 18.5, 19.5, 20.5 and 21.5 (term = 22 days). We obtained body weight (BW) and photographed the diaphragm area (DA), hernia area (HA) and subsequent calculated the HA/DA ratio in N+ group. Samples of Diaphragm muscle were processed for histological staining with H/E and immunohistochemistry (IHQ) for myosin. The fetuses of N- and N+ groups had decreased BW and DA compared to EC and OO groups (p < 0.001). HA was decreased on GD 18.5 compared to 21.5 (p < 0.001) and the HA/DA ratio showed no difference. IHQ showed decreased expression of myosin in nitrofen groups. CDH induced by nitrofen model contributes to the understanding of muscularization in the formation of the diaphragm where the myosin expression is decreased.

G.P.2.06 Diaphragm muscle weakness in an experimental intensive care unit model

Diaphragm muscle dysfunction contributing to weaning failure in critically ill patients remains unclear. It may not fully depend on ventilator-induced modifications. Sepsis and administration of pharmacological agents such as corticosteroids and neuromuscular blocking agents may be involved. The objectives of this study were to examine to which extent sepsis, corticosteroid treatment and neuromuscular blocking agent administration aggravate ventilator-related diaphragm dysfunction using a unique piglet intensive care unit model. Piglets were randomly sham-operated, or for five days, mechanically ventilated, mechanically ventilated together with an endotoxin-induced sepsis, mechanically ventilated together with corticosteroid treatment, mechanically ventilated together with neuromuscular blocking agent administration, or mechanically ventilated together with sepsis, corticosteroid and neuromuscular blocking agent. On day 5, muscle fibers were isolated from diaphragm biopsies and frozen or membrane-permeabilized. The cross-sectional area and maximal force normalized to cross-sectional area were evaluated at the single muscle fiber level. Myosin heavy chain and actin protein mRNA expression were assessed in diaphragm muscle samples. Results show (i) a decrease in fiber maximal force normalized to cross-sectional area coupled with a reduction in myosin heavy chain mRNA level in all mechanically ventilated piglets; (ii) a decrease in fiber size in piglets that were mechanically ventilated in combination with sepsis, corticosteroid and neuromuscular blocking agent. In conclusion, five days mechanical ventilation had a significant negative effect on the force generating capacity of individual diaphragm muscle fibers. Sepsis, corticosteroid or neuromuscular blocking agent had no additional on force generation or muscle size, but both force normalized to fiber cross-sectional area and fiber size decreased significantly when all were combined with mechanical ventilation.

Inotropic Effects of the Channel Blocker 3,4-Diaminopyridine: Differential Responses of Rat Soleus and Extensor Digitorum Longus

The K+ channel blocker 3,4-diaminopyrindine (DAP) increases diaphragm force, use of which could potentially improve muscle performance during functional neuromuscular stimulation. To determine the extent of hindlimb muscle force augmentation, and delineate whether DAP effects vary in muscles comprised of mainly slow versus fast fibers, rat soleus, extensor digitorum longus (EDL) and diaphragm muscle samples were studied in vitro. DAP increased force of all three muscles, but at high concentrations the force increases were transient and were followed by declines in force below baseline. The maximum DAP-induced twitch force increase was smaller for soleus (38 +/-7%) than both EDL (94+/-12%) (P < 0.05) and diaphragm (93+/-13%) (P < 0.01). During fatigue-inducing 20 Hz stimulation (tested at an intermediate DAP concentration), force of soleus muscle remained significantly elevated by DAP for the entire testing period, force of DAP-treated EDL muscle rapidly declined to values in untreated muscle, and force of DAP-treated diaphragm had an intermediate force-time profile. Muscles varied in extent to which isometric contractile kinetics were altered by DAP. Thus, the K+ channel blocker DAP improves contractile performance of limb muscles, but the profile of improvement is distinct between the soleus and EDL muscles.

Prevalence of exposure to Salmonella spp in finishing swine marketed in Iowa

To describe the prevalence of antibodies against Salmonella spp in swine marketed in Iowa. Swine marketed by 1,044 low-volume producers and 45 high-volume producers. Samples of diaphragm muscle collected from swine carcasses were tested by an indirect ELISA based on lipopolysaccharides from Salmonella spp, in particular Salmonella serovar Typhimurium. Prevalence of positive results for antibodies against Salmonella spp for carcasses, lots, and swine for each producer was determined. Producer-level seroprevalence was used to classify swine from producers as having negligible, low, moderate, or widespread evidence of previous or historical exposure to Salmonella spp. From low-volume producers, 23,609 of 25,478 (92.7%; 95% confidence interval [CI], 92.4% to 92.9%) samples had negative results, and 1,863 (7.3%; 95% CI, 7.05% to 7.56%) had antibodies against Salmonella spp. Of the 6,299 lots of swine tested, 1,191 (18.9%) contained at least 1 sample with positive results. From high-volume producers, 203 of 2,486 (8.1%; 95% CI, 6.8% to 9.3%) samples had antibodies against Salmonella spp, and 124 of 629 lots had at least 1 sample with positive results for antibodies against Salmonella spp. Less than 10% of pigs marketed in Iowa are apparently exposed to Salmonella spp. Most swine marketed by low-volume producers had negligible or little evidence of exposure to Salmonella spp, whereas a higher percentage of swine marketed by high-volume producers had positive results when tested to detect antibodies against Salmonella spp.

Effects of dietary sunflower seed oil on rumen protozoa population and tissue concentration of conjugated linoleic acid in sheep

Two experiments with sheep were performed to test the efficacy of a linoleic acid-rich sunflower seed oil as a supplement to barley silage-based diet (6% of the dietary dry matter) to suppress protozoal numbers in the rumen and measure effects of the oil on the growth performance and tissue content of conjugated linoleic acid (CLA). In the first experiment, rumen contents were sampled (2 h after feeding) from the two treatment groups of five rumen cannulated sheep on each day 0–14, and on days 16, 19, 21, 28, 35, 42 and 49. The pH and protozoal counts were determined in each sample, while volatile fatty acids (VFA) and ammonia nitrogen were determined in samples from days 7, 14, 21 and 49. In the second experiment, nine lambs were fed each of the two diets for 168 days. Thereafter, the lambs were harvested and samples of diaphragm muscle, leg muscle, rib muscle, heart, liver, kidney and subcutaneous fat were analyzed for lipid and fatty acid concentrations. Feeding the oil supplement decreased ( P<0.05) the total protozoal numbers in rumen fluid samples from approximately 1,000,000 to less than 200,000 ml −1 within 6 days and maintained low numbers thereafter. This was associated with higher ( P<0.05) pH on days 9 to 12, 21, 35, 42 and 49 and lower ( P<0.05) concentrations of ammonia nitrogen and total VFA. However, average daily gain, daily dry matter intake, feed conversion ratio, carcass weight and clean fleece weight were not affected ( P>0.05) by feeding of oil. The CLA content was increased ( P<0.05) by dietary supplementation of the oil in tissue samples of diaphragm muscle (55%), leg muscle (37%), rib muscle (33%) and subcutaneous fat (33%). It was concluded that sunflower seed oil reduced rumen fauna and the C16:0 proportion of fat, while increasing C18:2 and CLA content in the muscle and fat tissues.