Effects of Intestinal Contents from Normal and Immunized Mice on Sporozoites of Eimeria falciformis1

The interaction of Eimeria falciformis sporozoites with the intestinal epithelium and with the intestinal contents from the cecum and colon of normal and specifically immunized mice was studied by light (LM) and scanning electron (SEM) microscopy. Fecal (FM) and enterocyte-associated (EAM) mucus were removed from the cecum and colon of normal mice and mice that had been immunized 1, 6, 12, or 20 days earlier with a series of oral inoculations of E. falciformis oocysts. Sporozoite-specific IgA, but neither IgM nor IgG, was detected by the immunofluorescent antibody test in FM and EAM from immunized mice. No sporozoite-specific immunoglobulin was detected in normal mice. When examined by LM, sporozoites exposed to all FM and EAM preparations exhibited greater motility and excystation from sporocysts. At 4 h after incubation in FM or EAM from normal or immune mice, about 10% of the sporozoites appeared damaged, being non-motile and non-refractile. Immune FM and EAM caused agglutination of sporozoites and sporocysts and oocyst walls of E. falciformis. but did not agglutinate those of E. ferrisi. Scanning electron microscopy of in vitro interactions between E. falciformis sporozoites and intestinal contents revealed that sporozoites exposed to immune EAM were coated with particulate material whereas those exposed to normal EAM were relatively clean. Sporozoites exposed to immune FM were usually embedded within the mucus whereas those exposed to normal FM were situated on top of the mucus. No significant differences occurred between the length/width (L/W) ratios of sporozoites incubated in normal FM and EAM or in PBS. Sporozoites exposed to immune FM had significantly greater L/W ratios than those exposed to normal FM whereas those exposed to immune EAM had significantly shorter L/W ratios than ones exposed to normal EAM. Few of the sporozoites observed on the luminal surface of the colon and cecum of normal mice were covered by mucus and none was altered in shape or showed pellicular damage. Only a few sporozoites were observed on the luminal surface of the colon and cecum of immunized mice. Most of these were covered by mucus and some exhibited pellicular alterations.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

Susceptibility of Germ-free Mice to Infectious Megaenteron

Germ-free (GF)-ICR mice were shown to be less susceptible to oral inoculation with a pathogenic strain of Escherichia coli (E. coli 0115a, c: K(B)) than GF-CF#1 mice. In GF-CF#1 mice a large number of organisms were recovered from the intestinal wall from the cecum to the rectum 3 to 7 days after inoculation. Unlike those in GF-CF#1 mice, lesions in GF-ICR mice were localized in a part of the cecum and organisms were recovered only from the cecal wall and rarely from organs other than those of the alimentary tract. In both strains of mice, however, organisms were recovered in large number from the intestinal contents. Histopathology and immunofluorescence revealed organisms closely attached to the surface of the cecum, colon and rectal epithelia in GF-CF#1 mice but only in a part of the cecal epithelium in GF-ICR mice. After being in contact with conventional CF#1 mice for 21 days and then inoculated orally with the pathogenic E. coli, ex-GF-CF#1 mice died within 14 days with severe intestinal lesions, but ex-GF-ICR mice survived without lesions.     

Cellular Aspects of the Resistance of Chickens to Eimeria tenella Infections*

SYNOPSIS. Sporozoites of Eimeria tenella were injected into the peritoneal cavity of normal chickens and chickens immunized against E. tenella. In some experiments normal scrum and serum from resistant chickens were injected prior to the injection of sporozoites. After 15 or 30 minute periods of intraperitoneal incubation, exudates were harvested and the occurrence of intracellular sporozoites was determined. Only macrophages and degranulated granulocytes were observed to contain sporozoites. There was no significant difference between the number of macrophages obtained from normal chickens (normal macrophages) which contained sporozoites and the number of macrophages obtained from immune chickens (immune macrophages) which contained sporozoites. Significantly fewer immune macrophages treated with immune serum contained sporozoites than untreated normal or immune cells, normal macrophages treated with either serum, or immune macrophages treated with normal scrum. Sporozoites in untreated normal macrophages did not appear to be harmed by the intracellular environment, based on structural observations. The majority of sporozoites in macrophages from all other groups were difficult to distinguish within the cytoplasm and were visibly distorted. It is hypothesized that the presence of fewer infected macrophages in exudates of immune chickens and serum-treated normal chickens was caused by an enhanced ability of these cells to destroy the parasite. Similar observations were noted in the case of sporozoites within degranulated granulocytes of experimental groups. The lack of understanding of the degranulation phenomenon makes it difficult to interpret these findings.     

Sensitivity and Specificity of the Indirect Fluorescent Antibody Test in the Study of Four Murine Coccidia1

The sensitivity and specificity of the indirect fluorescent antibody (IFA) test for the detection of serum antibodies were examined in mice that were infected with Eimeria falciformis, E. ferrisi, E. papillata, or E. vermiformis. For the study of each species, five groups of mice were given graded inoculation doses of 10, 10², 10³, 10⁴, or 10⁵ sporulated oocysts in a primary infection. The sixth group was infected with three sequential doses of 1.5 times 10³, 1.5 times 10⁴, and 1.5 times 10⁵ sporulated oocysts per mouse at two- to three-week intervals. All groups of infected mice developed serum antibodies. Sera were titrated by the IFA test with purified sporozoites. Strong fluorescence and high IFA titers were observed with homologous reactions mainly with the sera from mice infected with the higher inoculation dose levels in primary infections and from those given three sequential inoculation doses. Immunological cross reaction among the four species of Eimeria occurred at dilutions of 1:10 to 1:160. Very weak or no fluorescence of free sporozoites was observed with sera from noninfected mice, and there was no fluorescence of sporozoites contained in intact sporocysts.     

Rodent malaria: BCG-induced protection and immunosuppression.

One dose of 10(7) viable units of Mycobacterium bovis, strain BCG, protected a significant number of Swiss mice from a primary challenge with 10(4) thoracic sporozoites of Plasmodium berghei. Immunization with irradiated sporozoites induced greater protection than that observed in BCG-treated with BCG and surviving a primary sporozoite challenge were not protected from rechallenge, whereas mice immunized with irradiated sporozoites and surviving initial challenge of sporozoites were solidly immune to further challenge. Immunizing mice with BCG and irradiated sporozoites simultaneously resulted in a synergistic effect of increased protection against a primary challenge of sporozoites only if the two immunogens were administered on the same day and if the mice were challenged 1 to 3 days later. Mice given BCG and irradiated sporozoites and surviving a primary challenge of sporozoites were unable to survive rechallenge. BCG given to mice previously immunized with irradiated sporozoites suppressed their protective immunity against sporozoite challenge.     

Ultrastructural localization of antigenic sites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and Eimeria tenella by monoclonal IgG and IgM antibodies

Immunoelectron microscopy was used to study the localization of monoclonal IgG (13.9 and 15.84) and IgM (10.84) antibodies generated against Eimeria tenella sporozoites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and E. tenella. A uniform layer of ferritin was present on sporozoites of E. tenella fixed chemically before the addition of 10.84, 13.90, or 15.84 (called prefixed), whereas postfixed (fixed chemically after exposure to monoclonal antibody) sporozoites lacked ferritin, indicating that the latter had capped immune complexes. Patches of ferritin were present on prefixed and postfixed sporozoites of E. acervulina exposed to 15.84, indicating that immune complexes containing 15.84 were not capped. Sporocysts of E. tenella exposed to 10.84 had a uniform layer of ferritin on their outer surface; ferritin was localized in patches on those exposed to 13.90 or 15.84. In E. acervulina sporocysts exposed to 15.84, ferritin was widely scattered on the outer surface but formed a uniform layer on the inner surface of the sporocyst wall. Patches of ferritin occurred on the inner layer of the oocyst walls of E. tenella and E. acervulina exposed to 10.84, 13.90, or 15.84. These findings indicate the shared antigen detected by 15.84 differed in relative amount, spatial distribution, and structural location in sporozoites and sporocysts of E. acervulina and E. tenella.     

The demonstration of pre-hepatic immune response to Fasciola hepatica in the mouse.

Harness E., Hughes D. L. and Doy T. G. 1976. The demonstration of a pre-hepatic immune response to Fasciola hepatica in the mouse. International Journal for Parasitology6: 15–17. Two groups of mice were immunized with either one oral dose of 20 irradiated metacercariae per mouse or 2 such doses given 7 days apart. Twenty-one days after immunization these mice together with non immunized controls were orally dosed with 100 normal metacercariae. Two days later immature flukes were recovered from the peritoneal cavity and counted. The numbers of flukes recovered from both immunized groups of mice were significantly lower than those obtained from the controls; this is taken to indicate that a protective mechanism operates at the intestinal wall.     

B-lymphocyte responses in the large intestine and mesenteric lymph nodes of mice infected with Eimeria falciformis (Apicomplexa)

B-cell responses of 3 immunoglobulin isotypes (IgA, IgG, and IgM) were investigated in the large intestine and mesenteric lymph nodes (MLN) of naive or immune mice after inoculation of oocysts of Eimeria falciformis. Primary and anamnestic IgA and IgG lymphocyte responses to E. falciformis occurred in the large intestine of nonimmune and immune mice, respectively. IgA-containing lymphocytes (IgAc) were the largest population of responding B cells in the large intestine. In infected mice, IgAc accumulated in the apical portion of the lamina propria, whereas IgG-containing lymphocytes (IgGc) were more numerous at the base of the lamina propria. No significant increase in the number of IgM-containing lymphocytes (IgMc) was observed in the lamina propria of the large intestine. Primary but no anamnestic B-cell responses occurred in the MLN, and immune mice actually had reduced numbers of IgAc and IgGc in the MLN when compared with naive mice. IgGc were the largest population of responding B cells in the MLN. Thus, IgAc appear to accumulate preferentially at the site of parasite development, whereas IgGc are primarily localized deeper in the lamina propria of the large intestine and in the draining lymph nodes of mice infected with E. falciformis.     

Importance of cecum inClostridium botulinum colonization of mice and relationship of organism to large bowel

Germ-free mice, tested more than two weeks after removing their cecum, were at least 10⁴ times more resistant than controls to intestinal colonization byClostridium botulinum type A or B. Histologic examinations were done with the cecum and colon taken fromC. botulinum-monoassociated mice. Specimens from mice with severe botulism had normal appearance. In specimens from chronically monoassociated mice, the pathogen was in the lumen, but not attached to the intestine. Scanning electron microscopy suggested a possible association of the pathogen with the mucous gel lining.     

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.     

Interaction of Eimeria tenella with intestinal mucin in vitro

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.     

Experimental vaccination of chicks with Plasmodium gallinaceum sporozoites. I. Circumsporozoite proteins are expressed by sporozoites recovered from both salivary glands and midguts of mosquitoes

Immunogenicity of Plasmodium gallinaceum sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8-9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral lactoferrin treatment resolves amoebic intracecal infection in C3H/HeJ mice

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an illness that affects many people around the world. We have previously reported that lactoferrin is able to kill E. histolytica in in vitro cultures. The aim of the present study was to evaluate the therapeutic effect of orally administered bovine lactoferrin in the control of intestinal amoebiasis of susceptible C3H/HeJ mice. The results showed that 20 mg lactoferrin/kg orally administered each day for 1 week was able to eliminate the infection in 63% of the mice, since neither trophozoites nor evidence of epithelial damage and (or) swelling were found in tissue sections of the cecum. The rest of the treated animals (37%) showed a decrease in trophozoite numbers and mucus secreted to the lumen, as compared with untreated and infected mice (p < 0.05). By immunohistochemistry, the profile of secreted cytokines in the cecum revealed that infected but untreated animals showed a mixed Th1/regulatory cytokines profile, whereas the cecum of mice treated (cured) showed a Th2 cytokine profile (IL-4) and expression of the multifunctional IL-6. In addition, cytokines and increasing cecal production of total IgA antibodies were found associated with little inflammation and disease control observed in the cecum of lactoferrin-treated animals. These results suggest that oral administration of lactoferrin can control intestinal amoebic infection probably by killing amoebas or favoring their removal and reestablish the antiinflammatory intestinal environment.     

Intestinal goblet cell mucus release. II. In vivo stimulation by antigen in the immunized rat.

We previously reported that the infusion of certain soluble immune complexes stimulated mucus release from the rat small intestine in vivo. The present studies sought to evaluate the response of the intestine of normal and immunized rats to the infusion of antigen alone. One hour after the intraduodenal infusion of antigen, small intestinal washings were obtained and analyzed for the presence of 35S-labeled, high m.w. glycoprotein of goblet cell origin. The amount of goblet cell glycoprotein released was estimated from the radioactivity present in the void volume of a Sepharose 4B gel filtration column. The release of goblet cell mucus was enhanced by antigen stimulation in orally immunized animals. The discharge of goblet cell mucus was not increased after antigen infusion in animals immunized by the i.p. route despite the induction of high levels of serum antibody. The inability to demonstrate release of mucus after antigen challenge in systemically immunized rats suggests that the amount or the type(s) of antibody required at the mucosal surface is produced only after oral immunization.     

Hyperimmune bovine colostrum neutralizes Cryptosporidium sporozoites and protects mice against oocyst challenge

Activity of colostral whey, produced by a cow immunized with oocysts of Cryptosporidium parvum and found to provide prophylaxis against cryptosporidiosis in calves, was tested in 2 experiments. In one experiment BALB/c mice were given the immune whey (HW), whey from a nonimmunized cow (CW), or a balanced salt solution (HBSS) before, during, and after oral inoculation with oocysts of C. parvum. Significantly fewer (P less than 0.05) C. parvum were found in mice that received HW (undiluted, 1:20 or 1:50) than in those treated with similarly diluted CW or with HBSS. In the second experiment it was determined that protection was mediated by specific anti-sporozoite activity when significantly fewer (P less than 0.05) C. parvum were found in mice that received sporozoites treated with HW diluted 1:20 or 1:50 compared with mice that received sporozoites treated with similarly diluted CW or with HBSS.     

Capping of immune complexes by sporozoites of Eimeria tenella

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.     

Experimental murine model for autoimmune enterocolitis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant

An experimental model for autoimmune enterocolitis was produced in mice by repeated immunization of homologous colon extract together with Klebsiella 03 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes in the intestinal lesions were characterized by infiltration with polymorphonuclear leukocytes in the lamina propria, muscularis mucosae and submucosa of repeatedly immunized mice. No such intestinal lesions were produced in mice receiving injections of colon extract alone or KO3 LPS alone. Development of the autoantibody and delayed-type hypersensitivity against colon extract were found in mice immunized with the mixture of colon extract and KO3 LPS. Distinct positive staining was detected specifically on the columnar epithelium of villi. Sera from hyperimmunized mice defined organ-specific antigens present in the intestine. Therefore, it was suggested that the intestinal lesions might be caused by an autoimmune mechanism.     

溃疡性结肠炎小鼠肠道黏膜和盲肠内容物菌群差异比较

了解溃疡性结肠炎(UC)小鼠肠道黏膜菌群的构成及其与盲肠内容物菌群的差异, 探讨肠道菌群在UC小鼠发病中的作用。

12只C57BL/6N雄性小鼠随机分为正常组、治疗组和模型组, 每组4只。采用葡聚糖硫酸钠盐(DSS)进行造模, 采用柳氮磺胺吡啶灌胃进行治疗, 造模第8天处死全部小鼠。采用16S rRNA基因高通量测序法对肠道黏膜和盲肠内容物菌群进行测序分析。

UC小鼠肠道黏膜菌群与盲肠内容物菌群相比, OTU数量和多样性均降低; Beta多样性分析显示其黏膜和盲肠内容物菌群差异较正常小鼠更大。在门和属的分类水平上, UC小鼠黏膜菌群中变形菌门、疣微菌门、魏斯菌属和肠球菌属丰度较正常小鼠升高, 厚壁菌门和拟杆菌门丰度降低; 阿克曼菌属在UC小鼠黏膜和盲肠内容物菌群中增多, 颤螺菌属在治疗组小鼠黏膜和盲肠内容物菌群中均减少。此外, 黏膜菌群中脱铁杆菌门Mucispirillum菌属丰度较高; 盲肠内容物中拟杆菌属丰度较高。物种组成分析中, 颤螺菌属、Mucispirillum、志贺菌属、魏斯菌属和肠球菌属聚类在一起。

UC小鼠黏膜菌群与盲肠内容物菌群在群落组成、多样性、优势菌群等方面存在明显差异, 黏膜菌群在UC发病机制中可能发挥着主要的作用。

     

Experimental Vaccination of Chicks with Plasmodium gallinaceum Sporozoites. I. Circumsporozoite Proteins Are Expressed by Sporozoites Recovered from Both Salivary Glands and Midguts of Mosquitoes1

Immunogenicity of Plasmodium gallinaceum Sporozoites for chicks and their in vitro reactivity with normal and specific immune sera were studied. Two sporozoite populations recovered from experimentally infected Aedes fluviatilis were used: sporozoites from salivary glands and sporozoites from midgut oocysts. Populations seven to nine days old of sporozoites recovered from salivary glands were infective for all chicks until the chicks were three weeks old; however, sporozoites recovered from midguts containing oocysts infected these chicks only if isolated on days 8–9, but not on day 7 after the mosquitoes' infective blood meal. Infectivity of the sporozoites was lost after exposure to ultraviolet (UV) light (30 min) or X-rays (13 krad). Inactivated sporozoites from both sources proved highly immunogenic to chicks that were immunized by several intravenous or intramuscular injections. These parasites elicited a strong humoral immune response in the chicks, as measured by the circumsporozoite precipitation (CSP) reaction. The levels of the CSP antibodies were similar with sporozoites from both sources, there being no detectable differences in the percentage of reactive sporozoites or the intensity of the CSP reaction with sera containing antibodies to either sporozoites from salivary glands or sporozoites from oocysts. These results provide the first evidence that avian malaria sporozoites express the circumsporozoite protein that has been extensively characterized in mammalian malaria (rodent, simian, human sporozoites). Furthermore, we observed that the yields of sporozoites obtained from mosquito midguts, on days 8 and 9 of the P. gallinaceum infection, were at least twice as great as those obtained by salivary gland dissection, even 20 days after a blood meal. This is an advantage since obtaining the midguts is less tedious, as well as more efficient and faster.