Treatment of cryptosporidiosis with oral bovine transfer factor.

Cryptosporidia are intestinal protozoans long known to cause diarrhea in humans, especially those with acquired immune deficiency syndrome (AIDS). When transfer factor prepared from calves which possessed delayed-type hypersensitivity to Eimeria bovis was given to nonimmune calves and mice it conferred protection against clinical infection (coccidiosis). Recent studies with oral bovine transfer factor have shown that it can confer cell-mediated immunity to humans. Based on these findings we decided to treat eight AIDS patients suffering from Cryptosporidium-associated diarrhea with transfer factor prepared from calves immune to Cryptosporidium. Prior to treatment with transfer factor, three patients had been treated with spiramycin, one patient with alpha-difluoromethylornithine (DFMO), and one patient with furazolidone for greater than 1 month without clinical or laboratory improvement. Following administration of transfer factor, five or eight patients exhibited a decrease in the number of bowel movements and the development of formed stools. Cryptosporidium was eradicated from the stools of four patients but two of these patients subsequently relapsed and one patient continued to have diarrhea despite the absence of Cryptosporidium in the stool. One patient has been free of diarrhea and Cryptosporidium for 2 years after discontinuation of transfer factor therapy.

Dermal cellular responses of helminth-free and Ostertagia ostertagi-infected calves to intradermal injections of soluble extracts from O. ostertagi L3 larvae.

Twelve calves were raised helminth-free until 9 weeks of age when six were orally inoculated with 100,000 Ostertagia ostertagi infective stage larvae (L3). Three uninfected and three experimentally infected calves received intradermal injections of sterile saline and soluble larval extract (SLE) from O. ostertagi L3 with a protein concentration ranging from 1 to 200 micrograms ml-1. Biopsies were performed 48 h post-injection. A kinetic study was performed on the remaining six calves, three infected and three uninfected, using a 100 micrograms ml-1 concentration of SLE and taking biopsies 1, 4, 8, 12, 24, and 72 h post-injection at both the saline and SLE-injected sites. All calves had an immediate wheal and increase in skin thickness at the SLE-injected sites. The numbers of eosinophils infiltrating SLE-injected sites as compared to saline-injected sites were significant in both uninfected and infected calves, but the infected calves had significant numbers to a wider range of SLE concentrations and had significantly higher numbers than uninfected calves in the kinetic study. Infected calves also had significant numbers of basophils in the dose response study at concentrations of 5 and 100 micrograms ml-1 SLE. Neutrophil infiltration was similar in both groups and was significant at SLE-injected sites early in the kinetic study. Detectable mast cells were decreased in SLE-injected sites of infected animals and perivascular accumulation of mononuclear and some polymorphonuclear cells was observed in the deep dermis of infected animals.

Lymphocyte blastogenic responses of calves experimentally infected with Ostertagia ostertagi.

Twelve 9-week-old calves were divided into four groups; two groups were maintained helminth-free as controls and the other groups were given Ostertagia ostertagi infective-stage larvae (L3) orally. One group received 100,000 L3 as a single inoculum and the other group received L3 in increasing dosages at weekly intervals for 8 consecutive weeks. The blastogenic responses to concanavalin A (Con A), phytohemagglutinin (PHA), and a soluble larval antigen from O. ostertagi (SLA) of peripheral blood lymphocytes were evaluated using tritiated thymidine incorporation into DNA as a measure of blastogenesis. The responses to Con A of all infected calves were significantly depressed while the responses to PHA were not. SLA, at concentrations of 4 micrograms ml-1 and above, caused blastogenic activity in lymphocytes from uninfected calves. Using SLA at 1 microgram ml-1 in lymphocyte cultures supplemented with autologous serum, an antigen-induced blastogenic response was detected in calves receiving serial inoculations of L3.

Immunomodulation with killed Propionibacterium acnes in guinea pigs simultaneously vaccinated with Brucella abortus strain 19.

Immunomodulation with killed Propionibacterium acnes was attempted in guinea pigs simultaneously vaccinated with Brucella abortus strain 19. Two groups, each comprised of 9 guinea pigs, were injected by different routes (s.c. and or i.v.) with 1.4 mg of P. acnes and 5 X 10(8) CFU of B. abortus, S-19, while 3 other groups each received either P. acnes, B. abortus S-19, or saline (s.c.). The antibody titers to B. abortus measured at 6, 10 and 14 weeks after vaccination indicated no significant (P less than 0.01) response in the 2 groups immunopotentiated with P. acnes concurrent with B. abortus S-19 vaccination. The delayed hypersensitivity response to 3 Brucella antigens conducted 8 weeks after immunization did not show a significant difference between the B. abortus S-19 vaccinated group compared with the 2 groups immunopotentiated and vaccinated. However, the proliferative response of lymphocytes to the B. abortus soluble antigen diluted 1:100 indicated significantly enhanced blastogenesis in the (s.c.) immunopotentiated and immunized guinea pigs compared with the B. abortus S-19 vaccinated group. A slightly enhanced response was also observed in the group immunopotentiated (i.v.) and vaccinated (s.c.). The guinea pigs were challenged with B. abortus strain 2308 and necropsied 4 weeks later. The mean splenic CFU of the Brucella in the group immunopotentiated (i.v.) and vaccinated (s.c.) was significantly decreased when compared with the guinea pigs vaccinated with B. abortus S-19 alone. These findings indicated that P. acnes administered simultaneously with B. abortus S-19 vaccine was able to augment the immune response in guinea pigs. Immunomodulation as evidenced by enhanced clearance of B. abortus from the spleens of immunopotentiated animals was presumably brought about by activated macrophages or a T-cell mediated cytolytic mechanism or both.

Infectivity of Cryptosporidium sp isolated from wild mice for calves and mice.

A study was conducted to determine the incidence of cryptosporidiosis in wild mice (Mus musculus) and the infectivity of oocysts from their feces for susceptible calves. The presence of oocysts and the duration of shedding of oocysts in the feces were evaluated in 115 wild mice. Approximately 30% of the mice shed Cryptosporidium sp oocysts, without evidence of clinical infection; recurrence of oocyst shedding was found in about 50% of the mice. Oocysts from the feces of naturally infected mice were infective for calves and mice. Calves began shedding oocysts at 7 days and shed oocysts for about 10 days. Nonfatal, clinical cryptosporidiosis developed in 7 infected calves. The mice began shedding oocysts at 6 days and shed oocysts for 12 days. Fatalities or clinical infection did not develop in 5 infected mice. The results indicated that Cryptosporidium-infected wild mice may be a source of cryptosporidiosis in susceptible calves.

The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens.

The life cycle and morphology of a previously undescribed species of Cryptosporidium isolated from commercial broiler chickens is described. The prepatent period for Cryptosporidium baileyi n. sp. was three days post oral inoculation (PI) of oocysts, and the patent period was days 4-24 PI for chickens inoculated at two days of age and days 4-14 for chickens inoculated at one and six months of age. During the first three days PI, most developmental stages of C. baileyi were found in the microvillous region of enterocytes of the ileum and large intestine. By day 4 PI, most parasites occurred in enterocytes of the cloaca and bursa of Fabricius (BF). Mature Type I meronts with eight merozoites first appeared 12 h PI and measured 5.0 x 4.9 micrometers. Mature Type II meronts with four merozoites and a large granular residuum first appeared 48 h PI and measured 5.1 x 5.1 micrometers. Type III meronts with eight short merozoites and a large homogeneous residuum first appeared 72 h PI and measured 5.2 x 5.1 micrometers. Microgamonts (4.0 x 4.0 micrometers) produced approximately 16 microgametes that penetrated into macrogametes (4.7 x 4.7 micrometers). Macrogametes gave rise to two types of oocysts that sporulated within the host cells. Most were thick-walled oocysts (6.3 x 5.2 micrometers), the resistant forms that passed unaltered in the feces. Some were thin-walled oocysts whose wall (membrane) readily ruptured upon release from the host cell. Sporozoites from thin-walled oocysts were observed penetrating enterocytes in mucosal smears. The presence of thin-walled, autoinfective oocysts and the recycling of Type I meronts may explain why chickens develop heavy intestinal infections lasting up to 21 days. Oocysts of C. baileyi were inoculated orally into several animals to determine its host specificity. Cryptosporidium baileyi did not produce infections in suckling mice and goats or in two-day-old or two-week-old quail. One of six 10-day-old turkeys had small numbers of asexual stages only in the BF. Four of six one-day-old turkeys developed mild infections only in the BF, and sexual stages of the parasite were observed in only one of the four. All seven one-day-old ducks and seven two-day-old geese developed heavy infections only in the BF with all known developmental stages present.

Serum antibody response to soluble extract of the third-larval stage of Ostertagia ostertagi in cattle.

Serum IgG, IgM, and IgA antibody responses against L3 antigens of Ostertagia ostertagi were monitored by enzyme-linked immunosorbent assay (ELISA) after one, two or multiple sequential inoculations of this nematode in calves. Following the first infection, antibody levels did not change. After a second inoculation, IgG increased significantly (P less than 0.05) after 2 months. IgG was not significantly increased 1 month after challenge inoculation. IgM and IgA antibody levels did not change following the first or second inoculations of L3. IgG antibody levels rose only slightly following multiple sequential inoculations with infectious L3. Results indicate that calves with ostertagiasis have very weak serum antibody responses to L3, and these appear to be of little value in detection of the infection in these animals.

Fatal cryptosporidiosis in quail.

Young quails kept in wire-floored cages experienced high mortality, beginning at age 5 days, from diarrhea that was unresponsive to antibiotic therapy. At necropsy, the small intestine had clear fluid content and the cecum was distended by brown foamy fluid. Histopathologic findings in the small intestine were shortened villi with detached enterocytes at the tip. Cryptosporidium sp. (confirmed by electron microscopy) were numerous in the microvillous border. Neither bacterial nor viral pathogens were detected. No infection was established in day-old chickens gavaged with feces and intestinal contents from infected quails. Thorough cleansing of the cages followed by application of commercial bleach prevented recurrence.

Ostertagia ostertagi: excretory secretory chemotactic substance from infective larvae as cause of eosinophil locomotion.

The infective (third stage) larva of Ostertagia ostertagi produced an excretory secretory substance that is chemotactic for bovine eosinophils. The eosinophil chemotactic substance was present in supernatants of larval cultures within 6 hr of incubation in Eagle's Minimum Essential Medium containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. The substance responsible for eosinophil chemotaxis has activity in small amounts, a molecular weight (MW) greater than 2000, and is heat labile at 100 C. Bovine eosinophils appeared to have a receptor for the chemotactic excretory secretory substance which is either identical or structurally similar to the previously described eosinophil chemotactic substance present in soluble third stage larval extracts. The larval substance may cause eosinophil accumulation in the abomasal tissue of cattle with ostertagiasis.

Dermal responses to Ostertagia ostertagi in Ostertagia ostertagi- and Cooperia punctata-inoculated calves.

Calves harboring patent Ostertagia ostertagi or Cooperia punctata were given intradermal injections of O ostertagi 3rd-stage larval antigen. The initial injections were followed 30 days later by a 2nd series of injections. Skin thickness was measured at injection sites for 72 hours after injection. Selected injection sites including saline solution control sites were biopsied at 30 minutes, at 3, 24, 48, and 72 hours, and at 30 days after injection. After the 1st series of injections, there was a clear distinction in dermal reactions between O ostertagi-inoculated calves and C punctata-inoculated calves; after 24 hours, reactions were not seen in the C punctata-inoculated calves. Marked dermal reactions occurred in the O ostertagi-inoculated calves. The reactions at 30 minutes and 3 hours were characterized by slight-to-extensive infiltration of neutrophils and dermal edema. The 24-hour cellular reaction was principally due to neutrophil and eosinophil infiltration with edema and necrosis. Reactions at 48 to 72 hours were due to eosinophils and perivascular accumulations of macrophages and lymphocytes. Necrosis, neutrophils, and edema were present in foci where fragments of nematodes were located. On reinjection, a clear distinction in dermal reactions between calves was not seen based on the type of nematode infection. Thirty days after dermal inoculation, large nodules developed at the site of the initial antigen injection. The nodules were characterized by marked intradermal proliferation of lymphocytes in a follicular pattern with occasional macrophages and rare multinucleated giant cells.

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from sporozoites to oocysts in human embryonic lung cell culture.

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6-16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10-18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K2Cr2O7 at 25 degrees C and 37 degrees C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.

Complete development of Cryptosporidium in cell culture.

Protozoan parasites of the genus Cryptosporidium cause a short-term, flu-like, gastrointestinal illness in immunocompetent persons and severe, persistent, life-threatening diarrhea in immunodeficient individuals. No effective therapy is available for the treatment of cryptosporidiosis in the immunodeficient host. Complete development (from sporozoite to sporulated oocyst) of a human isolate of Cryptosporidium was achieved in cultured human fetal lung cells and primary chicken kidney and porcine kidney cells. The growth of this newly recognized zoonotic agent in cell culture now provides a means of studying its behavior, development, and metabolism, and a mechanism for evaluation of potentially useful therapeutic agents.

Lymphocyte reactivity to Ostertagia ostertagi L3 antigen in type I ostertagiasis.

To better understand the immune response of calves infected with Ostertagia ostertagi, studies were conducted to examine cell-mediated immune responses to L3 antigen and phytohemagglutinin (PHA) by lymphocyte reactivity assay. The L3 antigen was prepared by freeze-thawing and sonication of exsheathed O ostertagi L3. Antigen preparations contained 40 to 60 micrograms of protein/ml and no detectable endotoxin. Cell-mediated immune responses of peripheral blood lymphocytes were determined in 3 groups of 12 calves each: (i) calves given consecutive multiple dose inoculations with L3; (ii) noninfected controls. Consecutive multiple-dose-inoculated calves showed marked increases in stimulation indices (SI) to L3 antigen over the SI of naturally inoculated calves (56.5 and 25.8, respectively). Negative SI were obtained in calves of the noninoculated control group (-1.0). No significant difference (P greater than or equal to 0.05) in response to PHA was obtained between lymphocytes from calves inoculated with O ostertagi and lymphocytes obtained from noninoculated control calves. There was significant (P less than or equal to 0.01) agreement between positive SI and evidence of patent infection (development of type I ostertagiasis). Specificity of lymphocyte reactivity was determined, using lymphocytes from 4 O ostertagi- and 2 Cooperia punctata-infected calves after challenge inoculation. Positive SI to L3 antigen were obtained, using lymphocytes from either O ostertagi- or C punctata-infected calves, indicating that there may be a lack of genus specificity for O ostertagi L3 antigen in lymphocyte reactivity assay. Kinetic studies of lymphocyte reactivity to L3 antigen and PHA showed that responses were briefly suppressed to both of these stimuli in prepatent calves.(ABSTRACT TRUNCATED AT 250 WORDS)

A seroepidemiologic survey of antibody to bluetongue virus in Alabama cattle.

Bluetongue (BT) is an insect transmitted viral disease of sheep that often causes mild or inapparent disease but rarely causes severe disease in cattle. Until recently, bluetongue viral infection was believed to be more prevalent in the Western United States, as compared with other regions of the country. However, a national survey for bluetongue antibody and clinical evidence of the disease in the Southeastern United States prompted the present investigation that was designed to determine the serological prevalence of BT virus in Alabama cattle. Results of the study demonstrated that 16% of the samples collected from 1,500 cattle in 64 of the 67 counties were positive. The prevalence of positive cattle in the western part of the State was significantly higher (P less than .001) than the prevalence in the eastern half of the State. On a herd basis, 52% of all herds tested had positive animals. Results of this study suggest that bluetongue infection is more common in the Southeastern United States than previously suspected.