Nonenzymatic isolation of Trichinella pseudospiralis infective L1 larvae at pH 7.4

Over half of the number of Trichinella pseudospiralis infective L1 larvae recovered from host carcasses by pepsin-HCl digestion were isolated from homogenized carcasses incubated in HBSS. More worms isolated by the latter method were viable compared to those isolated by pepsin-HCl digestion. When host carcasses infected with T. pseudospiralis were diced into pieces and incubated in HBSS, 30% more worms were recovered than from homogenized carcasses incubated in HBSS as above, and the majority of worms acquired by the former method were viable. The infectivity of T. pseudospiralis infective L1 larvae isolated from homogenized muscle in HBSS was 3.9 times greater than that for larvae recovered from homogenized carcasses by pepsin-HCl digestion. Only 4% and 0.8% of the number of T. spiralis recovered from homogenized muscle by pepsin-HCl digestion were isolated from homogenized or diced muscle incubated in HBSS, respectively. Fewer T. spiralis isolated from homogenized tissue in HBSS were viable compared to those recovered from homogenized carcasses digested in pepsin-HCl or diced carcasses incubated in HBSS.     

Commercial blast-freezing of third-stage Anisakis simplex larvae encapsulated in salmon and rockfish

Sixty-four fish were blast-frozen to -35 C for 15 hr to determine the effects of commercial blast-freezing on the viability of third-stage larvae of Anisakis simplex encapsulated in the muscle and viscera of sockeye salmon (Oncorhynchus nerka) and canary rockfish (Sebastes pinniger). Parallel tests were conducted on larval nematodes in 16 whole (round) salmon, 16 dressed salmon (heads and viscera removed), and 32 whole (round) rockfish. After blast-freezing, 4 in-the-round salmon, 4 dressed salmon, and 8 in-the-round rockfish were examined at 1, 24, 48, and 72 hr. A total of 3,539 dead and 6 live larvae were collected from the fish tissues after standard enzymatic digestion. Salmon were infected with 1,245 of these larvae, and rockfish with 2,300. The 6 live worms, 2 from salmon and 4 from rockfish rounds, were recovered from muscle 1 hr after freezing; they were slightly motile and showed severe internal damage. No viable worms were found at or after 24 hr. The commercial blast-freezing process effectively killed larval nematodes in whole or dressed fish. Market-ready samples of previously blast-frozen silver salmon (O. kisutch) and chum salmon (O. keta) fillets and chum salmon steaks yielded no live worms, thereby confirming the efficacy of this process.     

Efficient polymerase chain reaction detection of the second internal transcribed spacer of mucosa-derived larvae is dependent on the larval extraction method

Methods for estimating abundance of arrested gastrointestinal larvae in large mammal hosts by digestion of the gastrointestinal mucosa are well established. The effects of digestion on the success of species identification using the polymerase chain reaction (PCR) are, however, unknown. In this study, the relationship between numerical recovery of arrested larvae and the success of PCR-typing for the second internal transcribed spacer of ribosomal genes was characterized. Fresh and prefrozen mucosa of 4 sheep yielded very similar rates of recovery and PCR detection. When sheep mucosa were digested with neutral N-acetyl cysteine, recovery increased, whereas PCR detection remained constant (60-80%) with digest duration (1-16 hr). In contrast, when sheep and Svalbard reindeer mucosa were digested with acid-pepsin, recovery increased, whereas PCR detection declined to 0 with digest duration. Thus, to optimize recovery and PCR analysis of arrested gastrointestinal nematode larvae, acid-pepsin digestion of 1-2 hr for PCR detection and 16 hr for recovery, or neutral N-acetyl cysteine digestion of 8-16 hr for both assays, should be used.     

Experimental Trichinella infection in seals

The susceptibility of seals to infection with Trichinella nativa and the cold tolerant characteristics of muscle larvae in seal meat were evaluated. Two grey seals, Halichoerus grypus, were inoculated with 5000 (100 larvae/kg) T. nativa larvae and two grey seals with 50000 (1000 larvae/kg). One seal from each dose group and two control seals were killed at 5 and 10 weeks post-inoculation (p.i.). At 5 weeks p.i., infection was established in both low and high dose seals with mean larval densities of 68 and 472 larvae per gram (lpg), respectively, using eight different muscles for analyses. At 10 weeks p.i., mean larval densities were 531 and 2649 lpg, respectively, suggesting an extended persistence of intestinal worms. In seals with high larval density infections, the distribution of larvae in various muscles was uniform, but in one seal with a low larval density infection, predilection sites of larvae included muscle groups with a relative high blood flow, i.e. diaphragm, intercostal and rear flipper muscles. Trichinella-specific antibody levels, as measured by ELISA, increased during the 10 week experimental period. Infected seal muscle was stored at 5, -5 and -18 degrees C for 1, 4 and 8 weeks. Muscle larvae released from stored seal muscle by artificial digestion were inoculated into mice to assess viability and infectivity. Larvae from seal muscle 10 weeks p.i. tolerated -18 degrees C for 8 weeks but larvae from seal muscle 5 weeks p.i. tolerated only 1 week at -18 degrees C, supporting the hypothesis that freeze tolerance increases with the age of the host-parasite tissue complex. The expressed susceptibility to infection, extended production of larvae, antibody response and freeze tolerance of T. nativa in seals are new findings from the first experimental Trichinella infection in any marine mammal and suggest that pinnipeds (phocids, otariiids or walrus) may acquire Trichinella infection by scavenging even small amounts of infected tissue left by hunters or predators.     

Trichinella spiralis: newborn larval migration route in rats reexamined

The route by which Trichinella spiralis newborn larvae migrate from the small intestine to striated muscle was studied in inbred AO and random-bred Sprague-Dawley rats. Newborn larvae were quantitatively recovered from the thoracic duct lymph, peritoneal cavity, and hepatic portal vein blood during the course of a primary infection with 4000 muscle larvae. The total recovery of newborn larvae assessed in this manner was compared with the number of muscle larvae in control rats receiving the same infection. In both strains of rats, most of the newborn larvae were recovered from hepatic portal vein blood, fewer than 3% of newborn larvae were recovered from the thoracic duct lymph and peritoneal cavity combined. Long-term drainage of thoracic duct lymph (greater than 24 hr) significantly increased newborn larval recovery over short-term drainage (less than 24 hr). We conclude that there are several natural pathways of newborn larval migration that result in muscle larval establishment. These include direct invasion of capillaries and lymphatics in the intestine as well as migration through the intestinal serosa to the peritoneal cavity. In both AO and Sprague-Dawley rats, greater than or equal to 97% of newborn larvae migrate via the hepatic portal vein blood to the general circulation.     

Host immunity against newborn Trichinella spiralis larvae of different ages

The infectivity of newborn Trichinella spiralis larvae of different ages was studied in normal rats. Newborn larvae collected after incubation of adult worms in vitro for 2, 12, or 24 hr were injected intravenously (i.v.) into normal AO rats in 3 separate recipient groups. All recipient rats developed strikingly similar numbers of muscle larvae 20 days later. The susceptibility to immunity by newborn larvae of different ages was also studied. No difference was found when degree of protection was compared by assessing muscle larvae burden or peritoneal anti-newborn larvae effects after injection of newborn larvae of different ages either i.v. or intraperitoneally into immunized recipient rats. We conclude that newborn larvae of any age up to 24 hr have similar infectivity in normal rats and are equally susceptible to anti-newborn larvae immunity in vivo.     

Trichinella spiralis: vascular recirculation and organ retention of newborn larvae in rats

The recirculation of Trichinella spiralis newborn larvae was studied in inbred AO rats. Newborn larvae collected after in vitro incubation of adult T. spiralis worms for 2 or 24 hr were injected into rats through the tail vein or hepatic portal vein. Blood samples from the femoral vein, hepatic portal vein, and abdominal aorta were collected at intervals from 1 min to 24 hr after larval injection. Newborn larvae of both ages (24 hr or 2 hr old) persisted in femoral vein blood for less than or equal to 5 hr after injection, but they could be detected in portal vein blood by 24 hr after injection. The injection of larvae into a tail vein or the portal vein did not influence the pattern of larval circulation, although there was a 1-5 min delay in newborn larval appearance time after injection into the portal vein. Transcapillary migration through tissue and back to the circulation was evident in the appearance of newborn larvae in the thoracic duct lymph up to 24 (occasionally 48) hr after tail vein injection of newborn larvae. During the course of a natural primary infection, no evidence for trapping of larvae in the mesenteric lymph node could be found despite direct larval migration through this organ. Injected newborn larvae were retained in the lungs, and small numbers could be recovered 24 hr after intravenous injection. We conclude that a proportion of newborn larvae recirculates within the vasculature for several hours; a smaller population extravasates but can reenter the circulatory system via the lymphatics. Furthermore, some newborn larvae are found in organs rich in capillaries up to 24 hr after their entry into the blood.     

Trichinella murrelli in scavenging mammals from south-central Wisconsin, USA

Tissues and serum from 59 raccoons (Procyon lotor), 42 coyotes (Canis latrans), and seven Striped Skunks (Mephitis mephitis) collected in Dane and Iowa Counties, Wisconsin, USA, between October 2005 and March 2006 were microscopically and serologically examined for the presence of Trichinella spp. Encapsulated larvae were found on compression slides prepared from tongue tissues from a few animals. Complete tissue digestion of tongues revealed that 19% of the raccoons, 26% of the coyotes, and none of the seven skunks tested were infected with Trichinella spp. Cats were subsequently experimentally infected by feeding them the raccoon tissues containing muscle larvae, and muscle larvae isolated from the collected tongues were experimentally transmitted to mice. Multiplex polymerase chain reaction analysis of the isolated muscle larvae demonstrated two distinct bands migrating at 127 base pairs (bp) and 316 bp in all samples, which together are diagnostic for Trichinella murrelli; the isolates were assigned Istituto Superiore di Sanita (ISS) codes ISS1656 through ISS1667, and ISS1708 through ISS1710 by the International Trichinella Reference Centre. These findings extend the geographic range of T. murrelli into Wisconsin, USA.     

Trichinella spiralis in the black bear (Ursus americanus) of Pennsylvania: distribution, prevalence and intensity of infection

Samples of tongue or diaphragm from 2,056 black bears harvested in Pennsylvania during the 1981-1983 hunting seasons were examined for larvae of Trichinella spiralis by peptic digestion. Sixteen males and 21 females were infected. The overall prevalence of infection was 1.8%. Infected animals were distributed widely throughout the range of the bear in Pennsylvania. In samples from infected bears, the geometric mean density of muscle larvae was 7.8 per g of tissue (LPG). There were neither sex- nor age-related differences in prevalence or intensity of infection. Virtually all bears harvested in Pennsylvania are consumed as food, which often is shared widely among hunters, their friends and relatives. Furthermore, high densities of larvae occurred in some bears (i.e., 300, 348, 465, 512, 555, and 912 LPG). Thus, a basis for potential, single-source outbreaks of severe human trichinosis exists.     

Detection of Trichinella spiralis in a horse during routine examination in Italy

Routine examination for Trichinella infection by artificial digestion of 5-g samples of muscle tissue revealed the presence of muscle larvae in one out of 28 borses imported from Romania to an abattoir in Italy. The parasite, identified as Trichinella spiralis by the polymerase chain reaction, showed a reproductive capacity index of 68 in Swiss mice. Light microscope examination of 200 nurse cell-larva complexes showed that 22% of them were calcified and that the capsules of the non-calcified nurse cells were 17.5–27.5 μm (s=22.67 μm) thick and had very few cellular infiltrates. The serum samples from the parasitologically positive horse and from three other horses of the same stock, from which Trichinella larvae were not recovered by digestion, showed a low level of positivity as determined by ELISA and Western blot analyses using a crude antigen, whereas negative results were observed in both tests when an excretory-secretory antigen was used. The results, together with data from the literature, suggest that the horse had acquired the infection 8–10 months previously and confirm earlier observations obtained from experimental infections, which showed that muscle worm burden and specific circulating antibodies were lost several months after infection.     

旋毛虫肌幼虫细胞传代培养及超微结构观察

消化、分离观察旋毛虫(Trichinella spiralis)肌幼虫,获得肌幼虫细胞,用含10%胎牛血清的RPMI-1640培养液培养原代细胞,胰酶(含0.02?TA)消化法进行传代,透射电镜观察培养细胞超微结构,用多重PCR鉴定培养细胞。结果表明,在培养24～72h原代细胞开始贴壁,7～8d形成单层细胞,细胞间融合现象不明显,10～12d传一代。透射电镜显示旋毛虫细胞核为椭圆形,核膜、核仁清晰,核内染色质较丰富,胞浆含丰富的线粒体。细胞主要有两种类型:椭圆形和多角形,以椭圆形为主。多重PCR扩增培养细胞DNA,可见1条与旋毛虫肌幼虫DNA扩增产物相同的条带(173bp)。结果表明,旋毛虫肌幼虫细胞可在含10%胎牛血清的RPMI-1640培养液中传代培养。     

Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasite's behavioral and nutritional status.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Ultrastructure of the mushroom body: digestion during metamorphosis of Utterbackia imbecillis (Bivalvia: Unionidae)

Abstract. Larvae of the freshwater mussel Utterbackia imbecillis metamorphose to juveniles either during their attachment to a host fish, or in vitro in a culture medium. This transformation includes degeneration of larval structures and development of the juvenile morphology. Early in metamorphosis the cells comprising the larval mantle enlarge and project into the mantle cavity, forming a structure referred to as the mushroom body. Its cells, which are ultrastructurally very similar to digestive cells of adult bivalves, are involved in pinocytosis or phagocytosis of the larval adductor muscle and of tissue from the host fish that is enclosed between the larval shells. Ingested material is passed from pinosomes to heterophagosomes which in turn fuse with heterolysosomes, where final degradation of ingested material occurs. Acid phosphatase activity was detected in heterophagosomes and heterolysosomes of all animals examined. In larvae that metamorphosed in vitro , the apical cytoplasm of the cells of the mushroom body, and the extracellular spaces among them, also exhibited acid phosphatase activity. Larvae reared on a host fish accumulated substantial deposits of lipids and glycogen within larval mantle cells during metamorphosis, whereas larvae reared in vitro did not. The larval mantle cells which constitute the mushroom body appear to be the primary sites of intracellular digestion of the larval adductor muscle and host tissue during metamorphosis.     

The impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata

Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0–7.5 larvae/cm². This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.     

Experimental trichinellosis in reindeer

Six female reindeer calves were inoculated intraruminally with various doses of Trichinella muscle larvae. Four calves were inoculated with T. nativa, receiving 15,000 (n = 1), 5,000 (1), and 2,500 (2) larvae each. Two calves were inoculated with 5,000 T. spiralis larvae each. Blood samples were collected twice per week for total white blood cell (WBC) and differential counts and for serology using enzyme-linked immunosorbent assay (ELISA) based on T. spiralis excretory-secretory antigen. On day 56, the calves were slaughtered and muscle samples were examined according to the standard digestion method for Trichinella larvae. Blood samples were also collected twice a week from 4 uninoculated, but otherwise similar, reindeer calves corralled separately. Both the total WBC and eosinophil counts of the inoculated animals were, on average, higher during the experimental period. All the inoculated calves seroconverted, showing an increase in the optical density (OD) in the ELISA starting between day 23 and day 27 postinoculation. Very few muscle larvae (<0.08 larvae/g [lpg]) were to be found from the animals inoculated with T. nativa, but about 4 and 6 lpg were recovered from the masseter muscles of those inoculated with T. spiralis.     

Ultrastructural effects of lethal freezing on brain,muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly,Eurosta solidaginis

In preparation for winter low temperatures, larvae of the gall fly, Eurosta solidaginis, accumulate the cryoprotectants glycerol, sorbitol, and trehalose. The fat body cells of these freeze-tolerant larvae can survive intracellular freezing to -80 degrees C for 48 h even though no whole larvae survive this treatment. We hypothesized that some other tissue was more susceptible to freezing and therefore may be responsible for larval death. This paper compares the ultrastructure of brain, muscle, and Malpighian tubules between non-lethally frozen and lethally frozen freeze-tolerant larvae. The nuclei of cortical brain cells from lethally frozen larvae exhibited clumped chromatin and nuclear membranes with occasional expansions or 'blebs' of the intermembranous space, while the cytoplasm contained swollen spheres of endoplasmic reticulum. In contrast, non-lethally frozen brain contained nuclei with evenly dispersed chromatin, smooth nuclear membranes and a cytoplasm free of swollen endoplasmic reticulum. Muscle tissue of lethally frozen larvae contained disrupted myofilaments surrounding the Z-line in comparison to non-lethally frozen muscle which had myofilaments extending all the way to the Z-line. Alterations of Malpighian tubule cells from lethally frozen larvae included an extracted cytoplasm with swollen and rounded mitochondria. In contrast, Malpighian tubule cells from non-lethally frozen larvae had a more concentrated cytoplasm with many rod-shaped mitochondria. Results show alterations to all three tissue types due to lethal freezing. The brain tissue contained the most observable alterations and therefore may be the most susceptible to lethal freeze damage.     

Evaluation of the single radial diffusion technique for detection of nuclear polyhedrosis virus (NPV) infection in Heliothis zea

The single radial diffusion test is an effective method for detection of nuclear polyhedrosis virus infection in Heliothis zea larvae. Virus antigens were detected in some instances 48 hr after the larvae were exposed to virus. Most larvae tested positively for virus antigens 72 hr after exposure to the virus. The tests could be read within 4 hr if the incubation temperature was 35°C, and within 24 hr at 22°C.     

Simultaneous isolation of preparative amounts of RNA and DNA from Trichinella spiralis by cesium trifluoroacetate isopycnic centrifugation

A rapid method for simultaneously banding preparative amounts of RNA and DNA from Trichinella spiralis muscle larvae by isopycnic centrifugation in cesium trifluoroacetate (CsTFA) is described. Larvae were homogenized in guanidinium isothiocyanate and the DNA, RNA, glycogen, and denatured protein components were isopycnically separated without prior purification. This procedure resulted in the isolation of nucleic acids suitable for molecular biological application. Agarose gel electrophoresis of gradient fractions indicated the separation of undegraded RNA and DNA where total RNA was of sufficient purity to efficiently direct in vitro translation of parasite protein and total DNA was greater than 20 kb in size and sensitive to restriction endonuclease digestion. Oligo (dT)-purified poly(A)+ mRNA was 3.6% of total RNA with greater than 18% conversion to cDNA.     

Detection of Trichinella spiralis, T. britovi and T. pseudospiralis in muscle tissue with real-time PCR

Infections caused by Trichinella species occur throughout the world in many wild and domestic animals resulting in trichinellosis in men. In Europe, domestic pigs are predominantly infected by three Trichinella species: T. spiralis, T. britovi and T. pseudospiralis. Present methods for detection of Trichinella spp. (compressorium method, artificial digestion) do not always sufficiently recognize Trichinella larvae and these techniques are labor-intensive, time consuming and do not differentiate isolates on the species level since there are no distinguishing morphological features. Additionally, conventional PCRs cannot quantify numbers of larvae in infectious material. In order to better meet these requirements, we developed a real-time PCR assay for the accurate, rapid and specific identification of the three common European species of the genus Trichinella. The assay targets the large subunit of the mitochondrial rRNA (rrnL) and enables sensitive determination and discrimination of larvae in muscle tissue samples. The real-time PCR assay was developed and validated using reference and field strains from T. spiralis, T. britovi and T. pseudospiralis. In the described real-time PCR assay, the melting points of specific amplificates were always discernable via the melting curve from melting points of unspecific amplificates. This is important for the methods workflow because only C(T) values connected with the additional melting curve analysis allow a distinction of the individual species with confidence. The sensitivity of the technique enabled detection down to 0.1 Trichinella larva per gram meat sample. High disruption levels of tissues by mincing generally resulted in higher sensitivities than protocols without mincing. With its short completion time as well as accurate and specific detection of selected species this assay could become a convenient tool for the fast detection of Trichinella larvae in meat.