- Email: [email protected]
Lack of acute pathogenicity and toxicity in mice of an isolate of Metarhizium anisopliae var. anisopliae from spittlebugs
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 65 (2006) 278–287 www.elsevier.com/locate/ecoenv
Lack of acute pathogenicity and toxicity in mice of an isolate of Metarhizium anisopliae var. anisopliae from spittlebugs C. Torielloa,, A. Pe´rez-Torresb, A. Burciaga-Dı´ azc, H. Navarro-Barrancoa, A. Pe´rez-Mejı´ aa, M. Lorenzana-Jime´nezd, T. Mierc a Departamento de Microbiologı´a y Parasitologı´a, Mexico Departamento de Biologı´a Celular y Tisular, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Mexico D. F. 04510, Mexico c Departamento El Hombre y su Ambiente, Divisio´n de Ciencias Biolo´gicas y de la Salud, Universidad Auto´noma Metropolitana-Xochimilco, Mexico, D. F. 04960, Mexico d Departamento de Farmacologı´a, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Mexico D. F. 04510, Mexico b
Received 1 September 2004; received in revised form 27 May 2005; accepted 6 July 2005 Available online 31 August 2005
Abstract A monospore strain of Metarhizium anisopliae var. anisopliae (EH-479/2), isolated in Mexico from Aeneolamia sp., was tested for oral acute intragastric pathogenicity and toxicity in CD-1 mice, including a thorough histological study. Animals were inoculated by gavage with one dose (108 conidia/animal) of viable (72 mice) and nonviable (24 mice) conidia and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. At the end of the study, no mice showed clinical symptoms of illness, and the animals’ mean weight corresponded to that of healthy adults. No inflammatory reactions were identified in analyzed organs, suggesting the nonimmunogenic status of this fungal strain. Evidence of fungal germination was noted in two lymph nodes and in liver and lung of one dead mouse, out of 72 viable-conidia treated mice. There was no evidence of toxicity symptoms in mice inoculated with nonviable conidia. The results obtained support the nonpathogenic and nontoxic status of this fungal strain when administered in a sole intragastric dose in mice. r 2005 Elsevier Inc. All rights reserved. Keywords: Aeneolamia sp.; Metarhizium anisopliae var. anisopliae; Mice; Pathogenicity; Safety; Toxicity
1. Introduction Metarhizium anisopliae (Metsch.) Sorokin var. anisopliae is an important fungal entomopathogen, distributed worldwide in the soil, exhibiting a wide range of insect host species. It was first described, under the name Entomophothora anisopliae, as a pathogen of the wheat cockchafer in 1879 by Metschnikoff and later as M. anisopliae by Sorokin in 1883 (Tulloch, 1976). In a taxonomic revision by Driver et al. (2000), the genus Metarhizium was reassessed using sequence data from the ITS and 28S rDNA D3 regions and RAPD-patterns, Corresponding author. Fax: (52 55) 5623 2355.
E-mail address: [email protected] (C. Toriello). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.07.016
revealing 10 distinct clades. M. anisopliae var. anisopliae corresponds to clade 9. This fungus has been deemed safe and considered an environmentally acceptable alternative to chemical pesticides (Domsch et al., 1980; Zimmermann, 1993). In recent years, it has been registered as a microbial agent or is under commercial development for the biological control of several pests (Butt et al., 2001). M. anisopliae has been isolated in Mexico from the white corn grub pest, Phyllophaga sp. (Coleoptera: Melolonthidae) (Villalobos, 1992), and from spittlebugs, Aeneolamia sp. and Prosapia simulans (Homoptera: Cercopidae), which attack particularly sugarcane, damaging approximately 20% of 415,000 ha of sugarcane plantations in Mexico (Berlanga-Padilla et al., 1997),
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
and other basic crops such as beans, rice, corn, sorghum, soybeans, and pastures. The spittlebug is a major pest of staple and industrial crops in Central and South America (Faria and Magalha˜es, 2001; Ferrer, 2001; Toriello et al., 1999a). The safety of M. anisopliae var. anisopliae has been established using mammal and vertebrate models such as birds, fishes, guinea pigs, mice, rabbits, rats and reptiles, mainly from 1968 to 1983 (Austwick, 1980; Zimmermann, 1993). Mammalian safety tests with rodents and rabbits using inhalation, subcutaneous, intraperitoneal, and intraocular exposure (Burges, 1981; El-Kadi et al., 1983; Saik et al., 1990; Shadduck et al., 1982; Toriello et al., 1999b) have not demonstrated adverse effects or infectivity. However, several cases of human infections by M. anisopliae have been reported in both immunocompetent and immunoincompetent individuals, with one fatality in an immunoincompetent child (Burgner et al., 1998), keratitis in a healthy man (Cepero de Garcı´ a et al., 1997), and two cases of sinusitis in immunocompetent hosts (Revankar et al., 1999). Another case in mammals involved a cat with invasive rhinitis due to M. anisopliae (Muir et al., 1998). The high phenotypic and genotypic variability among M. anisopliae strains from different geographical localities (Fegan et al., 1993), as indicated, among others, by highly variable toxicities (Goettel and Jaronski, 1997), as well as the large-scale use of this fungus in countries such as Brazil, where 100,000 ha of sugarcane are treated each year (Faria and Magalha˜es, 2001), call for a thorough assessment of strains to be released in the field. According to Goettel et al. (2001), the development and use of fungi as microbial agents for pest control require tests with mammal models to assess possible human and animal health risks. The main objective of the present study was to ascertain the safety of M. anisopliae var. anisopliae strain EH-479/2 isolated from the spittlebug (Aeneolamia sp.). A standard protocol (Environmental Protection Agency (EPA), 1996) was adopted to assess the acute oral toxicity and pathogenicity of viable and inactivated (heat-killed) conidial spores to CD-1 mice.
2. Materials and methods 2.1. Fungus The monospore virulent strain (EH-479/2) of M. anisopliae var. anisopliae has been isolated in Mexico from the spittlebug, Aeneolamia sp. Spores of the original strain MaMV were obtained from the National Centre for Biological Control (Centro Nacional de Referencia de Control Biolo´gico-CNRCB, Colima, Me´xico), and a single spore culture was prepared according to the method of Goettel and Inglis (1997).
279
The monospore culture was preserved in sterile water, in mineral oil, and in liquid nitrogen cryopreservation at –196 1C and deposited in the fungal collection of the Laboratory of Basic Mycology of the Department of Microbiology and Parasitology, Faculty of Medicine, Universidad Nacional Auto´noma de Me´xico (UNAM), Mexico. For the inoculum, a fungal suspension of 2 108 conidia/mL in 1% Tween 80 was prepared from 7 day-old potato dextrose agar (PDA, Bioxo´n, Mexico) cultures at 28 1C. Before animal inoculation, the conidial suspension was centrifuged and Tween 80, replaced by sterile physiological saline solution (PSS). 2.2. Animals and infection procedures The test was performed according to the Microbial Pesticide Test Guidelines of the US Environmental Protection Agency (EPA) (1996). The acute oral pathogenicity and toxicity test was chosen because it is mentioned as one of the tests to follow in the respective regulation of Mexico, Norma Oficial Mexicana, NOM114STPS-1994, published in the Official Diary of the Federation on January 30, 1996. A total of 114 young adult CD-1 mice (57 male and 57 female), weighing approximately 25–30 g, were used. The animals were free of parasites or pathogens, and females were nulliparous and nonpregnant. The animals were fed mouse chow (Purina de Me´xico, Mexico) and sterilized water ad libitum. Males and females were housed in separate Plexiglass cages (37 27 15 cm) with nesting material (wood shavings), six individuals were housed per cage. Animals were maintained in a 14/ 10 h light/dark cycle, in a room thermostatically maintained at 2472 1C and constant humidity throughout the study. Mice were fasted overnight prior to spore administration. After the spores had been administered, food was withheld for a further 3–4 h. The acute oral toxicity and pathogenicity test was performed over a 21day observation period. Necropsies were performed at 3, 10, 17, and 21 days after inoculation. The number of test animals differed among treatment groups. Altogether, 72 individuals were treated with viable and 24 individuals with nonviable conidia. Eighteen mice were in the control group. Of these, 12 individuals were treated with 0.5 mL of PSS (vehicle control) and six individuals were left untreated (untreated control). According to the EPA protocol, mice inoculated with viable (to evaluate fungal pathogenicity) and nonviable conidia (to evaluate toxic properties of the fungus) received 108 conidia per test animal, administered in a single intragastric oral dose by gavage (forcible feeding via a force-pump and a tube passed into the stomach). The group inoculated with nonviable conidia was dosed with inactivated (heat-killed) conidia. Inactivation was achieved by incubating the spores for 1 h at 60 1C, allowing reasonable maintenance of the
ARTICLE IN PRESS 280
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
structural integrity of the fungus. Viability of the fungal inoculum was processed prior to the infection procedure by germination percentage on PDA for 19 h at 28 1C and was always more than 97%. The nonviability of the deactivated spores was tested based on colony forming units (CFU) on PDA using the dilution plate technique, according to Goettel and Inglis (1997). During the necropsies, gross pathological changes were recorded in liver, spleen, kidney, brain, lung, and representative lymph nodes. To examine the persistence of the fungus in the animals, macerated fragments of their organs and blood fluids were plated on Sabouraud agar supplemented with 0.005% chloramphenicol and 0.04% cycloheximide (SABA) (Bioxo´n). The plates were incubated for 7 days at 28 1C. 2.3. Cleareance of the conidia Two grams of feces (wet weight) from the test animals were collected 24, 48, 96 and 120 h after the inoculation and examined for the presence of the inoculated conidia. The feces were collected from each cage housing six individuals from each group of animals. The feces were diluted in 1% Tween 80 and plated on SABA, and the plates were incubated for 7 days at 28 1C. Cultures were checked daily, and the CFU were determined per gram of feces. 2.4. Observation of animals
(enlarged axillary lymph nodes, splenomegaly, liver paleness) found in organs of viable and nonviable conidia treated mice were analyzed after arcsine transformation of percentages using independent samples in 2-tailed t-test (a ¼ 0:05)(SPSS Version 12, 2003).
3. Results 3.1. Clinical observations The clinical examination of the animals revealed no changes or alterations in any of the endpoints and characters studied. None of the animals showed discernible clinical symptoms of illness. However, one mouse treated with viable spores died 2 days after inoculation, i.e., before the first necropsy. Throughout the study, the behavior of test and control animals was similar, and a gradual increase in body weight was observed in all treatment groups (Fig. 1). The final weight at test termination was not significantly different among groups (F ¼ 1:310; df ¼ 2, 38; Po0:282). 3.2. Clearance of the fungus Viable conidia of M. anisopliae var. anisopliae were found only in the sample taken at 24 h post-inoculation. The density of spores was 6.3 104 CFU/g of feces. No
A careful clinical examination of all animals was made at least once daily throughout the experiment to detect morphological, physiological, or behavioral changes. Mainly, we searched for the presence of scabs, sores, or areas of hair loss; ruffled and dirty fur; discharge around the eyes, nose, mouth or anus; and signs of aggression or docility, paralysis, weakness, lameness, or hunched posture. Weights of individual animals were recorded shortly before the inoculation and weekly thereafter, as well as at the time of necropsies. 2.5. Histopathological analysis Fragments of the studied organs were fixed in buffered picric acid–formaldehyde (Zamboni and De Marino, 1965) for 24 h and washed with sodium phosphate buffer. Paraffin-embedded tissue sections were stained using periodic acid Schiff (PAS) and Grocott’s methenamine silver nitrate method for fungi (Grocott, 1955) and observed using a single-blinde method. 2.6. Data anaylisis and statistics All weight data were analyzed using analysis of variance (ANOVA). All data from gross pathology
Fig. 1. Development of body weight over time (weight gain) for each of three treatment groups: viable and nonviable conidia-inoculated animals and control mice.
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
viable conidia were isolated from feces of mice inoculated with heat-killed conidia and control animals. 3.3. Recovery of the fungus from the animals M. anisopliae var. anisopliae was isolated from 17 of 360 organs examined, corresponding to 10 of 72 mice inoculated with viable conidia. Blood samples were always negative (Table 1), but the fungus was isolated from liver, spleen, kidney, brain, and lung and two axillary lymph nodes. The organ with the highest proportion of positive tests was the liver (8.3% of all test animals in the viable conidia treatment group), followed by spleen, brain, and lung (4.2% each) and kidney (2.8%). In the other treatment groups, i.e., heatkilled conidia and control animals, all tests were negative. 3.4. Gross pathology Mice treated with viable conidia presented enlarged axillary lymph nodes, splenomegaly, and liver paleness in 9.7%, 12.5%, and 6.9%, respectively. Splenomegaly stayed constant, 4.2% on day 3 and 2.8% in all days from 10 to 21. Mice treated with heat-killed conidia presented discrete axillary lymphadenitis and liver paleness, in 8.3% of all test animals each. Splenomegaly was not observed in these animals (Table 1). Total gross pathology was not significantly different between viable and nonviable conidia-treated mice for enlarged axillary lymph nodes (F ¼ 1:575; t ¼ 1:214; df ¼ 4; Po0:292), splenomegaly (F ¼ 4:160; t ¼ 2:53; df ¼ 4; Po0:063), and liver paleness (F ¼ 6:447; t ¼ 0:166; df ¼ 4; Po0:876). Kidneys, brain, and lungs did not show evidence of macroscopic damage. 3.5. Histopathology The histopathological analysis showed few inflammatory infiltrates in organs from mice inoculated with
281
viable and heat-killed M. anisopliae var. anisopliae. Spleens displaying abundant red pulp with large and numerous megakaryocytes, surrounding a hyperplasic white pulp with some germinal centers, corresponded to viable conidia-treated animals with splenomegaly (9 out of 72). Most animals treated with viable and deactivated conidia had spleens with less enlarged red pulp and more visible splenic sinusoids, as well as loose cellularity of splenic cords, but the number and size of megakaryocytes were similar to that control spleens. Likewise, the white pulp did not differ markedly from that of control animals. The white pulp of fungal-treated groups was formed by periarteriolar lymphoid sheaths that rarely had germinal centers (Figs. 2a–f). The liver did not show notable histological differences between mice treated with viable and inactivated conidia. In both cases, Ku¨pffer’s cells increased in size, which was apparently due to the presence of PASpositive debris in the cytoplasm or the expression of an activated condition. Rarely, small circulating clusters of eosinophiles were observed in the lumen of hepatic sinusoids. Otherwise, livers of conidia-treated mice were similar to livers of control animals (Figs. 3a–c). Axillary lymphadenitis was observed in a few of the mice treated with viable and deactivated spores (9.7% and 8.3%, respectively). Lymph nodes showed a thick capsule and prominent lymphatic nodules in the outer cortex. Only in mice inoculated with viable conidia were numerous free conidia and conidia engulfed by phagocytes observed in the capsule, trabecules and inside of subcapsular and paratrabecular sinuses (Figs. 4a and b). Internalized conidia were morphologically intact, but many showed signs of degradation (Fig. 4c). Rarely, clusters of conidia were also observed in medullary cords and sinuses, and in the center of lymphatic nodules. In the lymph node of one animal we found germinating conidia and fungal hyphae (Fig. 4d). Frequently, an eosinophilic infiltrate was observed near conidia.
Table 1 Positive organ cultures (viable conidia treatment only) and gross pathology (viable and non-viable conidia treatments) in mice 3, 10, 17 and 21 days after inoculation with Metarhizium anisopliae var. anisopliae EH-479/2 Day
3 10 17 21 Total
Gross pathology (per mouse)
Positive cultures (organs/mice)a
Enlarged axillary lymph nodes
Splenomegaly
Liver paleness
L
S
K
B
Lu
Bl
V-C
NV-C
V-C
NV-C
V-C
NV-C
2/18 2/18 0/18 2/18 6/72
1/18 1/18 0/18 1/18 3/72
1/18 0/18 0/18 1/18 2/72
0/18 0/18 0/18 3/18 3/72
1/18 0/18 0/18 2/18 3/72
0/18 0/18 0/18 0/18 0/72
0/18 2/18 0/18 5/18 7/72
0/6 1/6 1/6 0/6 2/24
3/18 2/18 2/18 2/18 9/72
0/6 0/6 0/6 0/6 0/24
3/18 1/18 0/18 1/18 5/72
2/6 0/6 0/6 0/6 2/24
Note: All control animals showed negative cultures and no gross pathology. a Number of organs/number of mice inoculated with viable conidia. Abbreviations: L ¼ liver, S ¼ spleen, K ¼ kidney, B ¼ brain, Lu ¼ lung, Bl ¼ blood, V-C ¼ viable conidia, NV-C ¼ nonviable conidia.
ARTICLE IN PRESS 282
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
Fig. 2. Spleen from mice treated with viable (a,b) and nonviable conidia of Metarhizium anisopliae var. anisopliae (c,d), and from control mice (e,f). Note the presence of a germinal centre (arrows, a) associated with eccentrically located arteriole (arrowhead, a) in white pulp, and numerous and large megakaryocytes (arrows, b) within a hypercellular red pulp in viable conidia-treated mice with splenomegaly. The white pulp, consisting of central arterioles (arrowhead; c,e) surrounded by periarteriolar lymphoid sheaths (arrows; c,e), with few or no germinal centers was observed in mice treated with inactivated conidia and controls. The red pulp in these animals showed some megakaryocytes (arrows; d,f), visible splenic sinusoids, and more loose splenic cords (arrowheads; d,f). Similar findings were made in mice treated with viable conidia not showing splenomegaly. PAS staining. 400.
No histopathological changes were found in the central nervous system, lungs, or kidneys in any of the studied groups. Except for the findings in lymph nodes mentioned above, no histological evidence of circulating or extravascular fungal structures within interstitial
tissue or parenchyma of organs analyzed, was obtained at any time during the experiment. One mouse died 2 days after inoculation with viable spores. Kidneys, spleen, lymph nodes, and nervous system of this animal showed no macroscopic and
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
283
the fungus or elsewhere within the liver parenchyma. Germinated and free conidia were also found among microcirculating components in lungs, mainly within strongly dilated capillaries. The fungus was associated with microthrombi or emboli that occluded these vessels (Fig. 5e). Surprisingly, the lung parenchyma did not show any other alteration.
4. Discussion
Fig. 3. Liver from mice treated with viable conidia (a) or nonviable conidia of Metarhizium anisopliae var. anisopliae (b), and from control animals (c). Livers did not show notable structural differences. Rarely, fungal structures were identified as well as inflammatory infiltrates. However, many Ku¨pffer cells of viable conidia-treated mice contained magenta stained debris that probably corresponds to phagocytosed and destroyed fungi (arrow; a). Deposits of glycogen were partially preserved in cytoplasm of hepatocytes of control mice (arrowheads; c). PAS staining. 1000.
microscopic damage. However, congestion of liver sinusoids and many hepatocytes with vacuoles or empty cytoplasm were observed. Inside the hepatic sinusoids, evidence of fungal growth was observed (Figs. 5a–d). Remarkably, no inflammatory reaction was evident near
Our results show that most animals inoculated with viable and inactivated conidia (with the exception of one dead mouse at day two post inoculation) were capable of managing the fungal challenge, despite the high dose administered (108 conidia/animal). The mycological and histological results demonstrated that, while the fungus can persist temporarily in mice, no inflammatory acute reactions were observed in the organs of mice inoculated with viable or heatkilled fungus. Gross pathology did not reveal abnormalities associated with toxicity or fungal infection in tissue organs. The splenomegaly observed in some mice could be explained by a proliferative response of Tdependent areas secondary to the antigenic challenge represented by fungi. However, the rarity of germinal centers in mice treated with viable conidia suggests that this fungal strain might be a poor immunogen. Moreover, inactivated conidia probably lose immunogenicity since the white pulp in spleens of mice treated with nonviable spores show no notable differences from to control animals. The congestive or hypercellular red pulp in the spleens, including the increased size and number of megakaryocytes, speculatively, might have been induced by interleukins (IL) such as IL-3 and IL-6 that regulate the proliferation and differentiation of myeloid progenitor cells (Kierszenbaum, 2002). Particularly, mouse spleen is a target organ for the synergist action of IL-3 and IL-6 to induce marked megakaryocytopoiesis and to increase the size of megakaryocytes (Carrington et al., 1991; Ishibashi et al., 1989). These cytokines originate mainly from activated T-cells and macrophages (Feldmann, 1996). Another speculative but interesting explanation is that the same viable fungus can produce molecules with stimulatory effects on murine haematopoietic stem cells and megakaryocyte progenitors, as reported for bacterial polar glycopeptidolipids (Vincent-Naulleau et al., 1997). The observed lymphadenitis appeared to be a consequence of hyperplasia of lymphoid tissue in enlarged spleens. However, the presence of abundant conidia phagocytosed by macrophages and of eosinophilic infiltrates must be taken in account. Unlike other organs, viable germinated conidia were observed in only one lymph node (with the exception of germinated conidia in organs of the dead mouse). Their presence,
ARTICLE IN PRESS 284
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
Fig. 4. Lymph node from mice treated with Metarhizium anisopliae var. anisopliae viable fungus. Numerous conidia located in a thick capsule (rectangle; a) and inside of the subcapsular sinus (a). Note that the lymphatic nodule (ln; a) is free of fungal structures. Clusters of conidia were located in the cytoplasm of capsular and trabecular macrophages (arrows; b,c), which had a foamy aspect or contained degraded fungi, observed as PAS-positive debris (arrowhead; c). Evidence of fungal growth in the lymph node was obtained in the only dead mouse. In this case, numerous free conidia (black arrowhead; d) conidia chains (arrows; d), germinated conidia (curved arrow; d), and hyphae (white arrowhead; d) were observed, mainly in the cortex of the lymph node (d). Grocott staining, a,b,d; PAS staining, c. a ¼ 200; b,c ¼ 1000; d ¼ 400.
mainly inside the subcapsular, paratrabecular, and medullary sinuses, and more rarely in B-dependent areas, suggests that the fungus can reach the axillary lymph nodes through afferent lymphatic vessels and also via blood vessels. In view of the intragastric administration used in the present study, axillary lymphadenitis is unusual. Axillary lymph nodes drain lymph from mammary glands and regional skin but not from the upper digestive segment. However, clusters of circulating fungus might obstruct the lymph flow at the lymphatic trunk level, and then lymph reflux would appear to be feasible. The only histopathological image with a slight tissue reaction at day 10 postinoculation, of mice treated with viable and nonviable conidia, showed activated Ku¨pffer’s cells, with cytoplasmic debris, suggesting probable fungal phagocytosis. The fungus was able to penetrate the intestine and disseminate by hematogenous spread in the host, but the lack of germinating conidia in all observed histological images (with the exception of the
dead mouse and one lymph node) suggests that the fungus was not able to germinate and grow within any of the organs examined. The presence of fungi associated with microthrombi or emboli occluding microvasculature in the dead mouse suggests a disseminated intravascular coagulation event. This effect could have been lethal. Alternatively, the mouse might have been more susceptible that the other test animals due to a natural immunosuppression state. A study with the human pathogenic fungus, Histoplasma capsulatum, showed that high densities of yeast cells may attach to and agglutinate human erythrocytes (Taylor et al., 2004). The image (Fig. 4e) of fungal conidia associated to microthrombi or emboli suggests that a similar phenomenon might have caused the death of the mouse in our study. However, further assays would be necessary to validate this hypothesis. Most studies with M. anisopliae showed that this fungus is not pathogenic or toxic to mammals (El-Kadi et al., 1983; Jevanand and Kannan, 1995; Shadduck
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
285
Fig. 5. Histopathological findings in the only dead mouse 2 days after intragastric inoculation with the fungus Metarhizum anisopliae var. anisopliae. Blastospore-like structures (arrow; a,b), germinated conidium (arrow; c), and hyphal body (arrow; d) were identified in the hepatic parenchyma. Conidia (arrows; e) and germinated conidia (arrowhead; e) were also identified circulating inside some dilated blood capillaries or embedded in thrombi or emboli (outlined with dots; e) occluding the microvasculature. No inflammatory response in liver and lungs, associated with fungal structures, was observed. Normal aspect alveoli are shown at the top and bottom (asterisk; e). PAS staining. a–d ¼ 1000; e ¼ 200.
ARTICLE IN PRESS 286
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
et al., 1982; Toriello et al., 1999b; Tsai et al., 1994) and all agree with the nonpathogenic and nontoxic condition of this fungus. The entomopathogenic fungi generally have a narrow or delimited host range (Vinson, 1990), which reduces the risk of infection in nontarget organisms such as humans and other homoeothermic animals. However, poikilothermic animals may be at higher risk because of lower body temperature. Although M. anisopliae has rarely been isolated from humans or other mammals, several cases of mycoses in humans and animals have been reported (Burgner et al., 1998; Cepero de Garcı´ a et al., 1997; Muir et al., 1998; Revankar et al., 1999). Moreover, it is possible that chronic exposure to and inhalation of high densities of conidia during the mass-production or use of fungal spores for biological control could lead to allergic sensitization (Roberts and Humber, 1981; Ward et al., 2000), as confirmed for other fungi (Kurup and Banerjee, 2000). Testing the pathogenicity and toxicity in mammals of entomopathogens is one way to produce the data needed to address safety concerns when an entomopathogen is going to be mass-produced (Siegel, 1997). In view of the low number of human cases of M. anisopliae infections recorded over the last 100 years, the risk appears to be extremely low. Similarly to other nonhuman pathogens (e.g., Ustilago maydis, a plant pathogen; Greer, 1978), opportunistic infections would be confined to severely immunocompromised patients. These, however, are unlikely to be exposed to M. anisopliae. The three-tier mammalian safety testing scheme for entomopathogens published in 1981 by the World Health Organization (WHO) (Mammalian safety, 1981), subsequently adopted by the US EPA in 1982 (Pesticide Assessment Guidelines, 1982), considered that successful passage through Tier I led to a candidate entomopathogen being declared safe with no limitations. However, if difficulties arose in the first tier of tests, Tiers II and III provided the development of exposure data as well as longer-term-studies involving both single and multiple exposures (Siegel and Shadduck, 1990). The acute oral toxicity and pathogenicity assay used in our study is part of the first tier of shortterm tests. With only 1.3% mortality and sublethal effects in o30% (mostly o10%) of the test animals, further testing would not normally be required, and the fungal pest control agent would be considered a low risk.
5. Conclusions In the present study, less than 3% of all mice exposed to a high challenge concentration of viable conidia
showed clinical manifestations of disease or evidence of spore germination in organ tissues. The only casualty recorded was probably due to intravascular coagulation 2 days after intragastric inoculation of M. anisopliae var. anisopliae viable conidia. Our results provide further evidence that this entomopathogenic fungus is safe to mammals. In view of the few incidences of mycoses reported we would like to cite Burges (1981): a no-risk situation does not exist, certainly not with chemical pesticides, and even with biological agents, one cannot absolutely prove a negative. Registration of a chemical is essentially a statement of usage in which risks are acceptable, and the same must be applied to biological agents. Acknowledgments The authors are grateful for the technical collaboration of Vero´nica Rodrı´ guez, to Victor Herna´ndezVela´zquez and Ange´lica Berlanga (CNRCB-SAGARPA, Mexico) for the M. anisopliae (MaMV) original strain, to Ingrid Mascher for editorial assistance, and to Leticia Espinosa Mingo (Information and Documentation Section, UAM-X) for her support in this work’s documentation. This work was financed by the Consejo Nacional de Ciencia y Tecnologı´ a (CONACYT) of Mexico, Project G-31451-B. References Austwick, P.K.C., 1980. The pathogenic aspects of the use of fungi: The need for risk analysis and registration of fungi. In: Ludholm, B., Stackerud, M. (Eds.), Environmental Protection and Biological Forms of Control of Pest Organisms, vol. 31. Ecology Bulletin, Stockholm, pp. 91–102. Berlanga-Padilla, A.M., Herna´ndez-Vela´zquez, V.M., Garza, G., 1997. Metarhizium anisopliae (Metch) Sorokin para el control microbiano de la mosca pinta Aeneolamia spp. Memorias XX Congreso Nacional de Control Biolo´gico. Sociedad Mexicana de Control Biolo´gico, Me´xico, pp. 51-53. Burges, H.D., 1981. Safety, safety testing and quality control of microbial pesticides. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Diseases 1970–1980. Academic Press, London, pp. 737–767. Burgner, D., Eagles, G., Burgess, M., Procopis, P., Rogers, M., Muir, D., Pritchard, R., Hocking, A., Priest, M., 1998. Disseminated invasive infection due to Metarhizium anisopliae in an immunocompromised child. J. Clin. Microbiol. 36, 1146–1150. Butt, T.M., Jackson, C., Magan, N., 2001. Introduction—Fungal biological control agents: Problems and potential. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents. CAB International, pp. 1–9. Carrington, P.A., Hill, R.J., Stenberg, P.E., Levin, J., Corash, L., Schreurs, J., Baker, G., Levin, F.C., 1991. Multiple in vivo effects of interleukin-3 and interleukin-6 on murine megakaryocytopoiesis. Blood 77, 34–41. Cepero de Garcı´ a, M.C., Arboleda, M.L., Barraquer, F., Grose, E., 1997. Fungal keratitis caused by Metarhizium anisopliae var. anisopliae. J. Med. Vet. Mycol. 35, 361–363.
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287 Domsch, K.H., Gams, W., Anderson, T.H., 1980. Compendium of Soil Fungi. Academic Press, London, pp. 413–415. Driver, F., Milner, R.J., Trueman, J.W.H., 2000. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol. Res. 104, 134–150. El-Kadi, M.K., Xara´, L.S., De Matos, P.F., Da Rocha, J.V.N., De Oliveira, D.P., 1983. Effects of the entomopathogen Metarhizium anisopliae on guinea pigs and mice. Environ. Entomol. 12, 37–42. Environmental Protection Agency (EPA), 1996. http://www.epa.gov/ pesticides/biopesticides/guidelines/oppts_885_3050.htm of 26/09/ 01, decessed 26 September 2001. Faria, M.R.de., Magalha˜es, B.P., 2001. O uso de fungos entomopatogeˆnico no Brasil. Biotecnol. Cieˆnc. Desenvol. 22, 18–21. Fegan, M., Manners, J.M., Maclean, D.J., Irwin, J.A.G., Samuels, K.D.Z., Holdom, D.G., Li, D.P., 1993. Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. J. Gen. Microbiol. 139, 2075–2081. Feldmann, M., 1996. Cell cooperation in antibody response. In: Roitt, I., Brostoff, J., Male, D. (Eds.), Immunology. Mosby, Barcelona, pp. 8.1–81.6. Ferrer, F., 2001. Biological control of agricultural insect pests in Venezuela: advances, achievements, and future perspectives. Biocontrol News Inform. 22, 67N–74N. Goettel, M.S., Hajek, A.E., Siegel, J.P., Evans, H.C., 2001. Safety of fungal biocontrol agents. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents. CAB International, pp. 347–375. Goettel, M.S., Inglis, G.D., 1997. Fungi: Hyphomycetes. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, San Diego, pp. 213–249. Goettel, M.S., Jaronski, S.T., 1997. Safety and registration of microbial agents for control of grasshoppers and locusts, In: Goettel, M.S., Johnson, D.L. (Eds.), Microbial Control of Grasshoppers and Locusts. Mem. Entomol. Soc. Can. 171, 83–99. Greer, D.L., 1978. Basidiomycetes as agents of human infections: a review. Mycopathologia 65, 133–139. Grocott, R.G., 1955. A stain for fungi in tissue sections and smears using Gomori methenamine silver nitrate technique. Am. J. Clin. Pathol. 25, 975–979. Ishibashi, T., Kimura, H., Shikama, Y., Uchida, T., Kariyone, S., Hirano, T., Kishimoto, T., Takatsuki, F., Akiyama, Y., 1989. Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood 74, 1241–1244. Jevanand, H.R., Kannan, N., 1995. Evaluation of Metarizium anisopliae as a biocontrol agent for coconut pest Oryctes rhinoceros and its mammalian toxicity test on rats. J. Ecotoxicol. Environ. Monitor. 5, 51–57. Kierszenbaum, A.L., 2002. Blood and hematopoiesis. In: Kierszenbaum, A.L. (Ed.), Histology and Cell Biology. An Introduction of Pathology. Mosby, St. Louis, pp. 147–174. Kurup, V.P., Banerjee, B., 2000. Fungal allergens and peptide epitopes. Peptides 21, 589–599. Mammalian safety of microbial agents for vector control: a WHO memorandum. (1981). Bull. W.H.O. 59, 857. Muir, D., Martin, P., Kendall, K., Malik, R., 1998. Invasive hyphomycotic rhinitis in a cat due to Metarhizium anisopliae. Med. Mycol. 36, 51–54. Pesticide Assessment Guidelines: Subdivision M, Biorational Pesticides, PB83-153965. 1982. US Environmental Protection Agency, Washington, DC, 1982.
287
Revankar, S.G., Sutton, D.A., Sanche, S.E., Rao, J., Zervos, M., Dashti, F., Rinald, M.G., 1999. Metarhizium anisopliae as a cause of sinusitis in immunocompetent hosts. J. Clin. Microbiol. 37, 195–198. Roberts, W., Humber, R.A., 1981. Entomogenous fungi. In: Cole, G.T., Kendrick, B. (Eds.), Biology of Conidial Fungi, vol. 2. Academic Press, New York, pp. 201–236. Saik, J.E., Lacey, L.A., Lacey, C.M., 1990. Safety of microbial insecticides to vertebrates—Domestic animals and wildlife. In: Laird, M., Lacey, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, Boca Raton, FL, pp. 115–134. Shadduck, J.A., Roberts, D.W., Lause, S., 1982. Mammalian safety tests of Metarhizium anisopliae: preliminary results. Environ. Entomol. 80, 189–192. Siegel, J.P., 1997. Testing the pathogenicity and infectivity of entomopathogens to mammals. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, San Diego, pp. 325–336. Siegel, J.P., Shadduck, J.A., 1990. Safety of microbial insecticides to vertebrates—Humans. In: Laird, M., Laird, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, Boca Raton, FL, pp. 101–113. Taylor, M.L., Duarte-Escalante, E., Pe´rez, A., Zenteno, E., Toriello, C., 2004. Histoplasma capsulatum yest cells attach and agglutinate human erythrocytes. Med. Micol. 42, 287–292. Toriello, C., Navarro-Barranco, H., Almengor, M.L., Mier, T., 1999a. Aplicacio´n de Metarhizium anisopliae (Metsch) Sorokin como bioinsecticida contra la mosca pinta (Homoptera: Cercopidae) en pastizales para ganado en Guatemala. Rev. Mex. Mic. 15, 119–121. Toriello, C., Navarro-Barranco, H., Martı´ nez-Jacobo, A., Mier, T., 1999b. Seguridad en ratones de Metarhizium anisopliae (Metsch) Sorokin aislado de Aeneolamia sp. (Homoptera: Cercopidae) en Me´xico. Rev. Mex. Mic. 15, 123–125. Tsai, S.F., Liao, J.W., Hung, W.K., Wang, S.H., 1994. Acute pulmonary toxicity, infectivity and pathogenicity of Metarhizium anisopliae on rats. Plant Prot. Bull. (Taichung) 36, 65–73. Tulloch, M., 1976. The genus Metarhizium. Trans. Br. Mycol. Soc. 66, 407–411. Villalobos, F.J., 1992. The potential of entomophatogens for the control of white grub pest of corn in Mexico. In: Jackson, T.A., Glare, T.R. (Eds.), Use of Pathogens in Scarab Pest Management. Intercept, Andover, pp. 253–260. Vincent-Naulleau, S., Thibault, D., Neway, T., Pilet, C., 1997. Stimulatory effects of polar glycopeptidolipids of Mycobacterium chelonae on murine haematopoietic stem cells and megakaryocyte progenitors. Res. Immunol. 148, 127–136. Vinson, S.B., 1990. Potential impact of microbial insecticides on beneficial arthropods in the terrestrial environment. In: Laird, M., Lacey, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, Boca Raton, FL, pp. 43–64. Ward, M.D., Madison, S.L., Sailstad, D.M., Gavettt, S.H., Selgrade, M.K., 2000. Allergen-triggered airway hyperresponsiveness and lung pathology in mice sensitized with the biopesticide Metarhizium anisopliae. Toxicology 143, 141–154. Zamboni, L., De Marino, C., 1965. Buffered picric acid–formaldehyde: a new rapid fixative for electron microscopy. J. Cell Biol. 35, 148A. Zimmermann, G., 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pesticide Sci. 37, 375–379.
Ecotoxicology and Environmental Safety 65 (2006) 278–287 www.elsevier.com/locate/ecoenv
Lack of acute pathogenicity and toxicity in mice of an isolate of Metarhizium anisopliae var. anisopliae from spittlebugs C. Torielloa,, A. Pe´rez-Torresb, A. Burciaga-Dı´ azc, H. Navarro-Barrancoa, A. Pe´rez-Mejı´ aa, M. Lorenzana-Jime´nezd, T. Mierc a Departamento de Microbiologı´a y Parasitologı´a, Mexico Departamento de Biologı´a Celular y Tisular, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Mexico D. F. 04510, Mexico c Departamento El Hombre y su Ambiente, Divisio´n de Ciencias Biolo´gicas y de la Salud, Universidad Auto´noma Metropolitana-Xochimilco, Mexico, D. F. 04960, Mexico d Departamento de Farmacologı´a, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Mexico D. F. 04510, Mexico b
Received 1 September 2004; received in revised form 27 May 2005; accepted 6 July 2005 Available online 31 August 2005
Abstract A monospore strain of Metarhizium anisopliae var. anisopliae (EH-479/2), isolated in Mexico from Aeneolamia sp., was tested for oral acute intragastric pathogenicity and toxicity in CD-1 mice, including a thorough histological study. Animals were inoculated by gavage with one dose (108 conidia/animal) of viable (72 mice) and nonviable (24 mice) conidia and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. At the end of the study, no mice showed clinical symptoms of illness, and the animals’ mean weight corresponded to that of healthy adults. No inflammatory reactions were identified in analyzed organs, suggesting the nonimmunogenic status of this fungal strain. Evidence of fungal germination was noted in two lymph nodes and in liver and lung of one dead mouse, out of 72 viable-conidia treated mice. There was no evidence of toxicity symptoms in mice inoculated with nonviable conidia. The results obtained support the nonpathogenic and nontoxic status of this fungal strain when administered in a sole intragastric dose in mice. r 2005 Elsevier Inc. All rights reserved. Keywords: Aeneolamia sp.; Metarhizium anisopliae var. anisopliae; Mice; Pathogenicity; Safety; Toxicity
1. Introduction Metarhizium anisopliae (Metsch.) Sorokin var. anisopliae is an important fungal entomopathogen, distributed worldwide in the soil, exhibiting a wide range of insect host species. It was first described, under the name Entomophothora anisopliae, as a pathogen of the wheat cockchafer in 1879 by Metschnikoff and later as M. anisopliae by Sorokin in 1883 (Tulloch, 1976). In a taxonomic revision by Driver et al. (2000), the genus Metarhizium was reassessed using sequence data from the ITS and 28S rDNA D3 regions and RAPD-patterns, Corresponding author. Fax: (52 55) 5623 2355.
E-mail address: [email protected] (C. Toriello). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.07.016
revealing 10 distinct clades. M. anisopliae var. anisopliae corresponds to clade 9. This fungus has been deemed safe and considered an environmentally acceptable alternative to chemical pesticides (Domsch et al., 1980; Zimmermann, 1993). In recent years, it has been registered as a microbial agent or is under commercial development for the biological control of several pests (Butt et al., 2001). M. anisopliae has been isolated in Mexico from the white corn grub pest, Phyllophaga sp. (Coleoptera: Melolonthidae) (Villalobos, 1992), and from spittlebugs, Aeneolamia sp. and Prosapia simulans (Homoptera: Cercopidae), which attack particularly sugarcane, damaging approximately 20% of 415,000 ha of sugarcane plantations in Mexico (Berlanga-Padilla et al., 1997),
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
and other basic crops such as beans, rice, corn, sorghum, soybeans, and pastures. The spittlebug is a major pest of staple and industrial crops in Central and South America (Faria and Magalha˜es, 2001; Ferrer, 2001; Toriello et al., 1999a). The safety of M. anisopliae var. anisopliae has been established using mammal and vertebrate models such as birds, fishes, guinea pigs, mice, rabbits, rats and reptiles, mainly from 1968 to 1983 (Austwick, 1980; Zimmermann, 1993). Mammalian safety tests with rodents and rabbits using inhalation, subcutaneous, intraperitoneal, and intraocular exposure (Burges, 1981; El-Kadi et al., 1983; Saik et al., 1990; Shadduck et al., 1982; Toriello et al., 1999b) have not demonstrated adverse effects or infectivity. However, several cases of human infections by M. anisopliae have been reported in both immunocompetent and immunoincompetent individuals, with one fatality in an immunoincompetent child (Burgner et al., 1998), keratitis in a healthy man (Cepero de Garcı´ a et al., 1997), and two cases of sinusitis in immunocompetent hosts (Revankar et al., 1999). Another case in mammals involved a cat with invasive rhinitis due to M. anisopliae (Muir et al., 1998). The high phenotypic and genotypic variability among M. anisopliae strains from different geographical localities (Fegan et al., 1993), as indicated, among others, by highly variable toxicities (Goettel and Jaronski, 1997), as well as the large-scale use of this fungus in countries such as Brazil, where 100,000 ha of sugarcane are treated each year (Faria and Magalha˜es, 2001), call for a thorough assessment of strains to be released in the field. According to Goettel et al. (2001), the development and use of fungi as microbial agents for pest control require tests with mammal models to assess possible human and animal health risks. The main objective of the present study was to ascertain the safety of M. anisopliae var. anisopliae strain EH-479/2 isolated from the spittlebug (Aeneolamia sp.). A standard protocol (Environmental Protection Agency (EPA), 1996) was adopted to assess the acute oral toxicity and pathogenicity of viable and inactivated (heat-killed) conidial spores to CD-1 mice.
2. Materials and methods 2.1. Fungus The monospore virulent strain (EH-479/2) of M. anisopliae var. anisopliae has been isolated in Mexico from the spittlebug, Aeneolamia sp. Spores of the original strain MaMV were obtained from the National Centre for Biological Control (Centro Nacional de Referencia de Control Biolo´gico-CNRCB, Colima, Me´xico), and a single spore culture was prepared according to the method of Goettel and Inglis (1997).
279
The monospore culture was preserved in sterile water, in mineral oil, and in liquid nitrogen cryopreservation at –196 1C and deposited in the fungal collection of the Laboratory of Basic Mycology of the Department of Microbiology and Parasitology, Faculty of Medicine, Universidad Nacional Auto´noma de Me´xico (UNAM), Mexico. For the inoculum, a fungal suspension of 2 108 conidia/mL in 1% Tween 80 was prepared from 7 day-old potato dextrose agar (PDA, Bioxo´n, Mexico) cultures at 28 1C. Before animal inoculation, the conidial suspension was centrifuged and Tween 80, replaced by sterile physiological saline solution (PSS). 2.2. Animals and infection procedures The test was performed according to the Microbial Pesticide Test Guidelines of the US Environmental Protection Agency (EPA) (1996). The acute oral pathogenicity and toxicity test was chosen because it is mentioned as one of the tests to follow in the respective regulation of Mexico, Norma Oficial Mexicana, NOM114STPS-1994, published in the Official Diary of the Federation on January 30, 1996. A total of 114 young adult CD-1 mice (57 male and 57 female), weighing approximately 25–30 g, were used. The animals were free of parasites or pathogens, and females were nulliparous and nonpregnant. The animals were fed mouse chow (Purina de Me´xico, Mexico) and sterilized water ad libitum. Males and females were housed in separate Plexiglass cages (37 27 15 cm) with nesting material (wood shavings), six individuals were housed per cage. Animals were maintained in a 14/ 10 h light/dark cycle, in a room thermostatically maintained at 2472 1C and constant humidity throughout the study. Mice were fasted overnight prior to spore administration. After the spores had been administered, food was withheld for a further 3–4 h. The acute oral toxicity and pathogenicity test was performed over a 21day observation period. Necropsies were performed at 3, 10, 17, and 21 days after inoculation. The number of test animals differed among treatment groups. Altogether, 72 individuals were treated with viable and 24 individuals with nonviable conidia. Eighteen mice were in the control group. Of these, 12 individuals were treated with 0.5 mL of PSS (vehicle control) and six individuals were left untreated (untreated control). According to the EPA protocol, mice inoculated with viable (to evaluate fungal pathogenicity) and nonviable conidia (to evaluate toxic properties of the fungus) received 108 conidia per test animal, administered in a single intragastric oral dose by gavage (forcible feeding via a force-pump and a tube passed into the stomach). The group inoculated with nonviable conidia was dosed with inactivated (heat-killed) conidia. Inactivation was achieved by incubating the spores for 1 h at 60 1C, allowing reasonable maintenance of the
ARTICLE IN PRESS 280
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
structural integrity of the fungus. Viability of the fungal inoculum was processed prior to the infection procedure by germination percentage on PDA for 19 h at 28 1C and was always more than 97%. The nonviability of the deactivated spores was tested based on colony forming units (CFU) on PDA using the dilution plate technique, according to Goettel and Inglis (1997). During the necropsies, gross pathological changes were recorded in liver, spleen, kidney, brain, lung, and representative lymph nodes. To examine the persistence of the fungus in the animals, macerated fragments of their organs and blood fluids were plated on Sabouraud agar supplemented with 0.005% chloramphenicol and 0.04% cycloheximide (SABA) (Bioxo´n). The plates were incubated for 7 days at 28 1C. 2.3. Cleareance of the conidia Two grams of feces (wet weight) from the test animals were collected 24, 48, 96 and 120 h after the inoculation and examined for the presence of the inoculated conidia. The feces were collected from each cage housing six individuals from each group of animals. The feces were diluted in 1% Tween 80 and plated on SABA, and the plates were incubated for 7 days at 28 1C. Cultures were checked daily, and the CFU were determined per gram of feces. 2.4. Observation of animals
(enlarged axillary lymph nodes, splenomegaly, liver paleness) found in organs of viable and nonviable conidia treated mice were analyzed after arcsine transformation of percentages using independent samples in 2-tailed t-test (a ¼ 0:05)(SPSS Version 12, 2003).
3. Results 3.1. Clinical observations The clinical examination of the animals revealed no changes or alterations in any of the endpoints and characters studied. None of the animals showed discernible clinical symptoms of illness. However, one mouse treated with viable spores died 2 days after inoculation, i.e., before the first necropsy. Throughout the study, the behavior of test and control animals was similar, and a gradual increase in body weight was observed in all treatment groups (Fig. 1). The final weight at test termination was not significantly different among groups (F ¼ 1:310; df ¼ 2, 38; Po0:282). 3.2. Clearance of the fungus Viable conidia of M. anisopliae var. anisopliae were found only in the sample taken at 24 h post-inoculation. The density of spores was 6.3 104 CFU/g of feces. No
A careful clinical examination of all animals was made at least once daily throughout the experiment to detect morphological, physiological, or behavioral changes. Mainly, we searched for the presence of scabs, sores, or areas of hair loss; ruffled and dirty fur; discharge around the eyes, nose, mouth or anus; and signs of aggression or docility, paralysis, weakness, lameness, or hunched posture. Weights of individual animals were recorded shortly before the inoculation and weekly thereafter, as well as at the time of necropsies. 2.5. Histopathological analysis Fragments of the studied organs were fixed in buffered picric acid–formaldehyde (Zamboni and De Marino, 1965) for 24 h and washed with sodium phosphate buffer. Paraffin-embedded tissue sections were stained using periodic acid Schiff (PAS) and Grocott’s methenamine silver nitrate method for fungi (Grocott, 1955) and observed using a single-blinde method. 2.6. Data anaylisis and statistics All weight data were analyzed using analysis of variance (ANOVA). All data from gross pathology
Fig. 1. Development of body weight over time (weight gain) for each of three treatment groups: viable and nonviable conidia-inoculated animals and control mice.
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
viable conidia were isolated from feces of mice inoculated with heat-killed conidia and control animals. 3.3. Recovery of the fungus from the animals M. anisopliae var. anisopliae was isolated from 17 of 360 organs examined, corresponding to 10 of 72 mice inoculated with viable conidia. Blood samples were always negative (Table 1), but the fungus was isolated from liver, spleen, kidney, brain, and lung and two axillary lymph nodes. The organ with the highest proportion of positive tests was the liver (8.3% of all test animals in the viable conidia treatment group), followed by spleen, brain, and lung (4.2% each) and kidney (2.8%). In the other treatment groups, i.e., heatkilled conidia and control animals, all tests were negative. 3.4. Gross pathology Mice treated with viable conidia presented enlarged axillary lymph nodes, splenomegaly, and liver paleness in 9.7%, 12.5%, and 6.9%, respectively. Splenomegaly stayed constant, 4.2% on day 3 and 2.8% in all days from 10 to 21. Mice treated with heat-killed conidia presented discrete axillary lymphadenitis and liver paleness, in 8.3% of all test animals each. Splenomegaly was not observed in these animals (Table 1). Total gross pathology was not significantly different between viable and nonviable conidia-treated mice for enlarged axillary lymph nodes (F ¼ 1:575; t ¼ 1:214; df ¼ 4; Po0:292), splenomegaly (F ¼ 4:160; t ¼ 2:53; df ¼ 4; Po0:063), and liver paleness (F ¼ 6:447; t ¼ 0:166; df ¼ 4; Po0:876). Kidneys, brain, and lungs did not show evidence of macroscopic damage. 3.5. Histopathology The histopathological analysis showed few inflammatory infiltrates in organs from mice inoculated with
281
viable and heat-killed M. anisopliae var. anisopliae. Spleens displaying abundant red pulp with large and numerous megakaryocytes, surrounding a hyperplasic white pulp with some germinal centers, corresponded to viable conidia-treated animals with splenomegaly (9 out of 72). Most animals treated with viable and deactivated conidia had spleens with less enlarged red pulp and more visible splenic sinusoids, as well as loose cellularity of splenic cords, but the number and size of megakaryocytes were similar to that control spleens. Likewise, the white pulp did not differ markedly from that of control animals. The white pulp of fungal-treated groups was formed by periarteriolar lymphoid sheaths that rarely had germinal centers (Figs. 2a–f). The liver did not show notable histological differences between mice treated with viable and inactivated conidia. In both cases, Ku¨pffer’s cells increased in size, which was apparently due to the presence of PASpositive debris in the cytoplasm or the expression of an activated condition. Rarely, small circulating clusters of eosinophiles were observed in the lumen of hepatic sinusoids. Otherwise, livers of conidia-treated mice were similar to livers of control animals (Figs. 3a–c). Axillary lymphadenitis was observed in a few of the mice treated with viable and deactivated spores (9.7% and 8.3%, respectively). Lymph nodes showed a thick capsule and prominent lymphatic nodules in the outer cortex. Only in mice inoculated with viable conidia were numerous free conidia and conidia engulfed by phagocytes observed in the capsule, trabecules and inside of subcapsular and paratrabecular sinuses (Figs. 4a and b). Internalized conidia were morphologically intact, but many showed signs of degradation (Fig. 4c). Rarely, clusters of conidia were also observed in medullary cords and sinuses, and in the center of lymphatic nodules. In the lymph node of one animal we found germinating conidia and fungal hyphae (Fig. 4d). Frequently, an eosinophilic infiltrate was observed near conidia.
Table 1 Positive organ cultures (viable conidia treatment only) and gross pathology (viable and non-viable conidia treatments) in mice 3, 10, 17 and 21 days after inoculation with Metarhizium anisopliae var. anisopliae EH-479/2 Day
3 10 17 21 Total
Gross pathology (per mouse)
Positive cultures (organs/mice)a
Enlarged axillary lymph nodes
Splenomegaly
Liver paleness
L
S
K
B
Lu
Bl
V-C
NV-C
V-C
NV-C
V-C
NV-C
2/18 2/18 0/18 2/18 6/72
1/18 1/18 0/18 1/18 3/72
1/18 0/18 0/18 1/18 2/72
0/18 0/18 0/18 3/18 3/72
1/18 0/18 0/18 2/18 3/72
0/18 0/18 0/18 0/18 0/72
0/18 2/18 0/18 5/18 7/72
0/6 1/6 1/6 0/6 2/24
3/18 2/18 2/18 2/18 9/72
0/6 0/6 0/6 0/6 0/24
3/18 1/18 0/18 1/18 5/72
2/6 0/6 0/6 0/6 2/24
Note: All control animals showed negative cultures and no gross pathology. a Number of organs/number of mice inoculated with viable conidia. Abbreviations: L ¼ liver, S ¼ spleen, K ¼ kidney, B ¼ brain, Lu ¼ lung, Bl ¼ blood, V-C ¼ viable conidia, NV-C ¼ nonviable conidia.
ARTICLE IN PRESS 282
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
Fig. 2. Spleen from mice treated with viable (a,b) and nonviable conidia of Metarhizium anisopliae var. anisopliae (c,d), and from control mice (e,f). Note the presence of a germinal centre (arrows, a) associated with eccentrically located arteriole (arrowhead, a) in white pulp, and numerous and large megakaryocytes (arrows, b) within a hypercellular red pulp in viable conidia-treated mice with splenomegaly. The white pulp, consisting of central arterioles (arrowhead; c,e) surrounded by periarteriolar lymphoid sheaths (arrows; c,e), with few or no germinal centers was observed in mice treated with inactivated conidia and controls. The red pulp in these animals showed some megakaryocytes (arrows; d,f), visible splenic sinusoids, and more loose splenic cords (arrowheads; d,f). Similar findings were made in mice treated with viable conidia not showing splenomegaly. PAS staining. 400.
No histopathological changes were found in the central nervous system, lungs, or kidneys in any of the studied groups. Except for the findings in lymph nodes mentioned above, no histological evidence of circulating or extravascular fungal structures within interstitial
tissue or parenchyma of organs analyzed, was obtained at any time during the experiment. One mouse died 2 days after inoculation with viable spores. Kidneys, spleen, lymph nodes, and nervous system of this animal showed no macroscopic and
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
283
the fungus or elsewhere within the liver parenchyma. Germinated and free conidia were also found among microcirculating components in lungs, mainly within strongly dilated capillaries. The fungus was associated with microthrombi or emboli that occluded these vessels (Fig. 5e). Surprisingly, the lung parenchyma did not show any other alteration.
4. Discussion
Fig. 3. Liver from mice treated with viable conidia (a) or nonviable conidia of Metarhizium anisopliae var. anisopliae (b), and from control animals (c). Livers did not show notable structural differences. Rarely, fungal structures were identified as well as inflammatory infiltrates. However, many Ku¨pffer cells of viable conidia-treated mice contained magenta stained debris that probably corresponds to phagocytosed and destroyed fungi (arrow; a). Deposits of glycogen were partially preserved in cytoplasm of hepatocytes of control mice (arrowheads; c). PAS staining. 1000.
microscopic damage. However, congestion of liver sinusoids and many hepatocytes with vacuoles or empty cytoplasm were observed. Inside the hepatic sinusoids, evidence of fungal growth was observed (Figs. 5a–d). Remarkably, no inflammatory reaction was evident near
Our results show that most animals inoculated with viable and inactivated conidia (with the exception of one dead mouse at day two post inoculation) were capable of managing the fungal challenge, despite the high dose administered (108 conidia/animal). The mycological and histological results demonstrated that, while the fungus can persist temporarily in mice, no inflammatory acute reactions were observed in the organs of mice inoculated with viable or heatkilled fungus. Gross pathology did not reveal abnormalities associated with toxicity or fungal infection in tissue organs. The splenomegaly observed in some mice could be explained by a proliferative response of Tdependent areas secondary to the antigenic challenge represented by fungi. However, the rarity of germinal centers in mice treated with viable conidia suggests that this fungal strain might be a poor immunogen. Moreover, inactivated conidia probably lose immunogenicity since the white pulp in spleens of mice treated with nonviable spores show no notable differences from to control animals. The congestive or hypercellular red pulp in the spleens, including the increased size and number of megakaryocytes, speculatively, might have been induced by interleukins (IL) such as IL-3 and IL-6 that regulate the proliferation and differentiation of myeloid progenitor cells (Kierszenbaum, 2002). Particularly, mouse spleen is a target organ for the synergist action of IL-3 and IL-6 to induce marked megakaryocytopoiesis and to increase the size of megakaryocytes (Carrington et al., 1991; Ishibashi et al., 1989). These cytokines originate mainly from activated T-cells and macrophages (Feldmann, 1996). Another speculative but interesting explanation is that the same viable fungus can produce molecules with stimulatory effects on murine haematopoietic stem cells and megakaryocyte progenitors, as reported for bacterial polar glycopeptidolipids (Vincent-Naulleau et al., 1997). The observed lymphadenitis appeared to be a consequence of hyperplasia of lymphoid tissue in enlarged spleens. However, the presence of abundant conidia phagocytosed by macrophages and of eosinophilic infiltrates must be taken in account. Unlike other organs, viable germinated conidia were observed in only one lymph node (with the exception of germinated conidia in organs of the dead mouse). Their presence,
ARTICLE IN PRESS 284
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
Fig. 4. Lymph node from mice treated with Metarhizium anisopliae var. anisopliae viable fungus. Numerous conidia located in a thick capsule (rectangle; a) and inside of the subcapsular sinus (a). Note that the lymphatic nodule (ln; a) is free of fungal structures. Clusters of conidia were located in the cytoplasm of capsular and trabecular macrophages (arrows; b,c), which had a foamy aspect or contained degraded fungi, observed as PAS-positive debris (arrowhead; c). Evidence of fungal growth in the lymph node was obtained in the only dead mouse. In this case, numerous free conidia (black arrowhead; d) conidia chains (arrows; d), germinated conidia (curved arrow; d), and hyphae (white arrowhead; d) were observed, mainly in the cortex of the lymph node (d). Grocott staining, a,b,d; PAS staining, c. a ¼ 200; b,c ¼ 1000; d ¼ 400.
mainly inside the subcapsular, paratrabecular, and medullary sinuses, and more rarely in B-dependent areas, suggests that the fungus can reach the axillary lymph nodes through afferent lymphatic vessels and also via blood vessels. In view of the intragastric administration used in the present study, axillary lymphadenitis is unusual. Axillary lymph nodes drain lymph from mammary glands and regional skin but not from the upper digestive segment. However, clusters of circulating fungus might obstruct the lymph flow at the lymphatic trunk level, and then lymph reflux would appear to be feasible. The only histopathological image with a slight tissue reaction at day 10 postinoculation, of mice treated with viable and nonviable conidia, showed activated Ku¨pffer’s cells, with cytoplasmic debris, suggesting probable fungal phagocytosis. The fungus was able to penetrate the intestine and disseminate by hematogenous spread in the host, but the lack of germinating conidia in all observed histological images (with the exception of the
dead mouse and one lymph node) suggests that the fungus was not able to germinate and grow within any of the organs examined. The presence of fungi associated with microthrombi or emboli occluding microvasculature in the dead mouse suggests a disseminated intravascular coagulation event. This effect could have been lethal. Alternatively, the mouse might have been more susceptible that the other test animals due to a natural immunosuppression state. A study with the human pathogenic fungus, Histoplasma capsulatum, showed that high densities of yeast cells may attach to and agglutinate human erythrocytes (Taylor et al., 2004). The image (Fig. 4e) of fungal conidia associated to microthrombi or emboli suggests that a similar phenomenon might have caused the death of the mouse in our study. However, further assays would be necessary to validate this hypothesis. Most studies with M. anisopliae showed that this fungus is not pathogenic or toxic to mammals (El-Kadi et al., 1983; Jevanand and Kannan, 1995; Shadduck
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
285
Fig. 5. Histopathological findings in the only dead mouse 2 days after intragastric inoculation with the fungus Metarhizum anisopliae var. anisopliae. Blastospore-like structures (arrow; a,b), germinated conidium (arrow; c), and hyphal body (arrow; d) were identified in the hepatic parenchyma. Conidia (arrows; e) and germinated conidia (arrowhead; e) were also identified circulating inside some dilated blood capillaries or embedded in thrombi or emboli (outlined with dots; e) occluding the microvasculature. No inflammatory response in liver and lungs, associated with fungal structures, was observed. Normal aspect alveoli are shown at the top and bottom (asterisk; e). PAS staining. a–d ¼ 1000; e ¼ 200.
ARTICLE IN PRESS 286
C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287
et al., 1982; Toriello et al., 1999b; Tsai et al., 1994) and all agree with the nonpathogenic and nontoxic condition of this fungus. The entomopathogenic fungi generally have a narrow or delimited host range (Vinson, 1990), which reduces the risk of infection in nontarget organisms such as humans and other homoeothermic animals. However, poikilothermic animals may be at higher risk because of lower body temperature. Although M. anisopliae has rarely been isolated from humans or other mammals, several cases of mycoses in humans and animals have been reported (Burgner et al., 1998; Cepero de Garcı´ a et al., 1997; Muir et al., 1998; Revankar et al., 1999). Moreover, it is possible that chronic exposure to and inhalation of high densities of conidia during the mass-production or use of fungal spores for biological control could lead to allergic sensitization (Roberts and Humber, 1981; Ward et al., 2000), as confirmed for other fungi (Kurup and Banerjee, 2000). Testing the pathogenicity and toxicity in mammals of entomopathogens is one way to produce the data needed to address safety concerns when an entomopathogen is going to be mass-produced (Siegel, 1997). In view of the low number of human cases of M. anisopliae infections recorded over the last 100 years, the risk appears to be extremely low. Similarly to other nonhuman pathogens (e.g., Ustilago maydis, a plant pathogen; Greer, 1978), opportunistic infections would be confined to severely immunocompromised patients. These, however, are unlikely to be exposed to M. anisopliae. The three-tier mammalian safety testing scheme for entomopathogens published in 1981 by the World Health Organization (WHO) (Mammalian safety, 1981), subsequently adopted by the US EPA in 1982 (Pesticide Assessment Guidelines, 1982), considered that successful passage through Tier I led to a candidate entomopathogen being declared safe with no limitations. However, if difficulties arose in the first tier of tests, Tiers II and III provided the development of exposure data as well as longer-term-studies involving both single and multiple exposures (Siegel and Shadduck, 1990). The acute oral toxicity and pathogenicity assay used in our study is part of the first tier of shortterm tests. With only 1.3% mortality and sublethal effects in o30% (mostly o10%) of the test animals, further testing would not normally be required, and the fungal pest control agent would be considered a low risk.
5. Conclusions In the present study, less than 3% of all mice exposed to a high challenge concentration of viable conidia
showed clinical manifestations of disease or evidence of spore germination in organ tissues. The only casualty recorded was probably due to intravascular coagulation 2 days after intragastric inoculation of M. anisopliae var. anisopliae viable conidia. Our results provide further evidence that this entomopathogenic fungus is safe to mammals. In view of the few incidences of mycoses reported we would like to cite Burges (1981): a no-risk situation does not exist, certainly not with chemical pesticides, and even with biological agents, one cannot absolutely prove a negative. Registration of a chemical is essentially a statement of usage in which risks are acceptable, and the same must be applied to biological agents. Acknowledgments The authors are grateful for the technical collaboration of Vero´nica Rodrı´ guez, to Victor Herna´ndezVela´zquez and Ange´lica Berlanga (CNRCB-SAGARPA, Mexico) for the M. anisopliae (MaMV) original strain, to Ingrid Mascher for editorial assistance, and to Leticia Espinosa Mingo (Information and Documentation Section, UAM-X) for her support in this work’s documentation. This work was financed by the Consejo Nacional de Ciencia y Tecnologı´ a (CONACYT) of Mexico, Project G-31451-B. References Austwick, P.K.C., 1980. The pathogenic aspects of the use of fungi: The need for risk analysis and registration of fungi. In: Ludholm, B., Stackerud, M. (Eds.), Environmental Protection and Biological Forms of Control of Pest Organisms, vol. 31. Ecology Bulletin, Stockholm, pp. 91–102. Berlanga-Padilla, A.M., Herna´ndez-Vela´zquez, V.M., Garza, G., 1997. Metarhizium anisopliae (Metch) Sorokin para el control microbiano de la mosca pinta Aeneolamia spp. Memorias XX Congreso Nacional de Control Biolo´gico. Sociedad Mexicana de Control Biolo´gico, Me´xico, pp. 51-53. Burges, H.D., 1981. Safety, safety testing and quality control of microbial pesticides. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Diseases 1970–1980. Academic Press, London, pp. 737–767. Burgner, D., Eagles, G., Burgess, M., Procopis, P., Rogers, M., Muir, D., Pritchard, R., Hocking, A., Priest, M., 1998. Disseminated invasive infection due to Metarhizium anisopliae in an immunocompromised child. J. Clin. Microbiol. 36, 1146–1150. Butt, T.M., Jackson, C., Magan, N., 2001. Introduction—Fungal biological control agents: Problems and potential. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents. CAB International, pp. 1–9. Carrington, P.A., Hill, R.J., Stenberg, P.E., Levin, J., Corash, L., Schreurs, J., Baker, G., Levin, F.C., 1991. Multiple in vivo effects of interleukin-3 and interleukin-6 on murine megakaryocytopoiesis. Blood 77, 34–41. Cepero de Garcı´ a, M.C., Arboleda, M.L., Barraquer, F., Grose, E., 1997. Fungal keratitis caused by Metarhizium anisopliae var. anisopliae. J. Med. Vet. Mycol. 35, 361–363.
ARTICLE IN PRESS C. Toriello et al. / Ecotoxicology and Environmental Safety 65 (2006) 278–287 Domsch, K.H., Gams, W., Anderson, T.H., 1980. Compendium of Soil Fungi. Academic Press, London, pp. 413–415. Driver, F., Milner, R.J., Trueman, J.W.H., 2000. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol. Res. 104, 134–150. El-Kadi, M.K., Xara´, L.S., De Matos, P.F., Da Rocha, J.V.N., De Oliveira, D.P., 1983. Effects of the entomopathogen Metarhizium anisopliae on guinea pigs and mice. Environ. Entomol. 12, 37–42. Environmental Protection Agency (EPA), 1996. http://www.epa.gov/ pesticides/biopesticides/guidelines/oppts_885_3050.htm of 26/09/ 01, decessed 26 September 2001. Faria, M.R.de., Magalha˜es, B.P., 2001. O uso de fungos entomopatogeˆnico no Brasil. Biotecnol. Cieˆnc. Desenvol. 22, 18–21. Fegan, M., Manners, J.M., Maclean, D.J., Irwin, J.A.G., Samuels, K.D.Z., Holdom, D.G., Li, D.P., 1993. Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. J. Gen. Microbiol. 139, 2075–2081. Feldmann, M., 1996. Cell cooperation in antibody response. In: Roitt, I., Brostoff, J., Male, D. (Eds.), Immunology. Mosby, Barcelona, pp. 8.1–81.6. Ferrer, F., 2001. Biological control of agricultural insect pests in Venezuela: advances, achievements, and future perspectives. Biocontrol News Inform. 22, 67N–74N. Goettel, M.S., Hajek, A.E., Siegel, J.P., Evans, H.C., 2001. Safety of fungal biocontrol agents. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents. CAB International, pp. 347–375. Goettel, M.S., Inglis, G.D., 1997. Fungi: Hyphomycetes. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, San Diego, pp. 213–249. Goettel, M.S., Jaronski, S.T., 1997. Safety and registration of microbial agents for control of grasshoppers and locusts, In: Goettel, M.S., Johnson, D.L. (Eds.), Microbial Control of Grasshoppers and Locusts. Mem. Entomol. Soc. Can. 171, 83–99. Greer, D.L., 1978. Basidiomycetes as agents of human infections: a review. Mycopathologia 65, 133–139. Grocott, R.G., 1955. A stain for fungi in tissue sections and smears using Gomori methenamine silver nitrate technique. Am. J. Clin. Pathol. 25, 975–979. Ishibashi, T., Kimura, H., Shikama, Y., Uchida, T., Kariyone, S., Hirano, T., Kishimoto, T., Takatsuki, F., Akiyama, Y., 1989. Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood 74, 1241–1244. Jevanand, H.R., Kannan, N., 1995. Evaluation of Metarizium anisopliae as a biocontrol agent for coconut pest Oryctes rhinoceros and its mammalian toxicity test on rats. J. Ecotoxicol. Environ. Monitor. 5, 51–57. Kierszenbaum, A.L., 2002. Blood and hematopoiesis. In: Kierszenbaum, A.L. (Ed.), Histology and Cell Biology. An Introduction of Pathology. Mosby, St. Louis, pp. 147–174. Kurup, V.P., Banerjee, B., 2000. Fungal allergens and peptide epitopes. Peptides 21, 589–599. Mammalian safety of microbial agents for vector control: a WHO memorandum. (1981). Bull. W.H.O. 59, 857. Muir, D., Martin, P., Kendall, K., Malik, R., 1998. Invasive hyphomycotic rhinitis in a cat due to Metarhizium anisopliae. Med. Mycol. 36, 51–54. Pesticide Assessment Guidelines: Subdivision M, Biorational Pesticides, PB83-153965. 1982. US Environmental Protection Agency, Washington, DC, 1982.
287
Revankar, S.G., Sutton, D.A., Sanche, S.E., Rao, J., Zervos, M., Dashti, F., Rinald, M.G., 1999. Metarhizium anisopliae as a cause of sinusitis in immunocompetent hosts. J. Clin. Microbiol. 37, 195–198. Roberts, W., Humber, R.A., 1981. Entomogenous fungi. In: Cole, G.T., Kendrick, B. (Eds.), Biology of Conidial Fungi, vol. 2. Academic Press, New York, pp. 201–236. Saik, J.E., Lacey, L.A., Lacey, C.M., 1990. Safety of microbial insecticides to vertebrates—Domestic animals and wildlife. In: Laird, M., Lacey, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, Boca Raton, FL, pp. 115–134. Shadduck, J.A., Roberts, D.W., Lause, S., 1982. Mammalian safety tests of Metarhizium anisopliae: preliminary results. Environ. Entomol. 80, 189–192. Siegel, J.P., 1997. Testing the pathogenicity and infectivity of entomopathogens to mammals. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathology. Academic Press, San Diego, pp. 325–336. Siegel, J.P., Shadduck, J.A., 1990. Safety of microbial insecticides to vertebrates—Humans. In: Laird, M., Laird, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, Boca Raton, FL, pp. 101–113. Taylor, M.L., Duarte-Escalante, E., Pe´rez, A., Zenteno, E., Toriello, C., 2004. Histoplasma capsulatum yest cells attach and agglutinate human erythrocytes. Med. Micol. 42, 287–292. Toriello, C., Navarro-Barranco, H., Almengor, M.L., Mier, T., 1999a. Aplicacio´n de Metarhizium anisopliae (Metsch) Sorokin como bioinsecticida contra la mosca pinta (Homoptera: Cercopidae) en pastizales para ganado en Guatemala. Rev. Mex. Mic. 15, 119–121. Toriello, C., Navarro-Barranco, H., Martı´ nez-Jacobo, A., Mier, T., 1999b. Seguridad en ratones de Metarhizium anisopliae (Metsch) Sorokin aislado de Aeneolamia sp. (Homoptera: Cercopidae) en Me´xico. Rev. Mex. Mic. 15, 123–125. Tsai, S.F., Liao, J.W., Hung, W.K., Wang, S.H., 1994. Acute pulmonary toxicity, infectivity and pathogenicity of Metarhizium anisopliae on rats. Plant Prot. Bull. (Taichung) 36, 65–73. Tulloch, M., 1976. The genus Metarhizium. Trans. Br. Mycol. Soc. 66, 407–411. Villalobos, F.J., 1992. The potential of entomophatogens for the control of white grub pest of corn in Mexico. In: Jackson, T.A., Glare, T.R. (Eds.), Use of Pathogens in Scarab Pest Management. Intercept, Andover, pp. 253–260. Vincent-Naulleau, S., Thibault, D., Neway, T., Pilet, C., 1997. Stimulatory effects of polar glycopeptidolipids of Mycobacterium chelonae on murine haematopoietic stem cells and megakaryocyte progenitors. Res. Immunol. 148, 127–136. Vinson, S.B., 1990. Potential impact of microbial insecticides on beneficial arthropods in the terrestrial environment. In: Laird, M., Lacey, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, Boca Raton, FL, pp. 43–64. Ward, M.D., Madison, S.L., Sailstad, D.M., Gavettt, S.H., Selgrade, M.K., 2000. Allergen-triggered airway hyperresponsiveness and lung pathology in mice sensitized with the biopesticide Metarhizium anisopliae. Toxicology 143, 141–154. Zamboni, L., De Marino, C., 1965. Buffered picric acid–formaldehyde: a new rapid fixative for electron microscopy. J. Cell Biol. 35, 148A. Zimmermann, G., 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pesticide Sci. 37, 375–379.