- Email: [email protected]
Epizootic cutaneous pythiosis in beef calves
Veterinary Microbiology 109 (2005) 121–128 www.elsevier.com/locate/vetmic
Epizootic cutaneous pythiosis in beef calves Rosa Cristina Pe´rez a, Juan J. Luis-Leo´n a,b, Jose´ Luis Vivas c, Leonel Mendoza d,* a
University of Carabobo, Faculty of Health Science Health, Department of Microbiology (Aragua), Venezuela b Center of Biomedical Research, University of Carabobo (BIOMED-UC), Venezuela c Laboratory Clinic Lugo ‘‘Delgado Launois’’, Maracay, Estado de Aragua, Venezuela d Medical Technology Program, Microbiology and Molecular Genetics, Michigan State University, 322 North Kedzie Hall, East Lansing, MI 48824-1031, USA Received 30 December 2004; received in revised form 16 April 2005; accepted 19 April 2005
Abstract Enzootic bovine granulomatosis (EBG) is a disease that affects beef calves in the flooded Savanna regions of Venezuela. Although Pythium insidiosum was originally the suspected etiologic agent, nothing was done to demonstrate its presence in the infected animals. The objective of this study was to investigate the etiologic agent causing cutaneous lesions in a group of 63 calves diagnosed with EBG. The collected samples were analyzed by histopathology, immunohistochemistry, serology, and mycological techniques. The clinical manifestations of BEG included ulcerated and swollen granulomatous lesions on the limbs of young calves. Histopathological studies revealed an eosinophilic inflammatory reaction with neutrophils, giant cells and branching hyphae with the Splendore–Hoeppli phenomenon. Immunofluorescence analysis identified the branching hyphae as elements of the fungal-like organism P. insidiosum. Wet mounts in 10% KOH revealed hyaline slender branching hyaline coenocytic hyphae, but cultures from those samples were negative. Anti-P. insidiosum immunoglobulins were also demonstrated in the collected sera of the affected bovines using latex agglutination, immunodiffusion, ELISA, and Western Blot. The histopathological, immunohistochemical, and serological tests showed for the first time that P. insidiosum could be involved in epizootic bovine pythiosis. # 2005 Elsevier B.V. All rights reserved. Keywords: Bovine pythiosis; Epizootic pythiosis; Pythium insidiosum
1. Introduction Pythiosis is a life threatening disease of the tropical, subtropical, and temperate areas of the * Corresponding author. Tel.: +1 517 353 7800; fax: +1 517 432 2006. E-mail address: [email protected] (L. Mendoza).
world. It is characterized by the development chronic lesions on the cutaneous and subcutaneous, intestinal, and bone tissues, as well as invasion of blood vessels in the infected animals and humans (Mendoza et al., 1996). The etiologic agent is the aquatic fungal-like striminipilan pathogen Pythium insidiosum. The infections are acquired when susceptible animals
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2005.04.020
122
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
and humans contact the infecting propagules of this pathogen through traumatic lesions in the enzootic areas. If the disease is not treated in the early stages, the infected hosts often die within weeks (Chaffin et al., 1995; Thomas and Lewis, 1998; Thianprasit, 1990). Chemotherapy using antifungal drugs has been difficult (Hutchins and Johnston, 1972; McMullan et al., 1977) and traditional treatments such as surgery and iodides produce limited results (Hutchins and Johnston, 1972; Miller, 1981; Thianprasit, 1990). More recently, immunotherapy has showed a promising new way to treat equines, dogs, and humans with the disease (Hensel et al., 2003; Mendoza et al., 2003; Thiatithanyanont et al., 1998; Wanachiwanawin et al., 2004). Pythiosis is more frequently diagnosed in equines and dogs (Alfaro and Mendoza, 1990; Chaffin et al., 1995; Thomas and Lewis, 1998) with few cases reported in bovines (Miller et al., 1985; Santurio et al., 1998), felines (Thomas and Lewis, 1998), ovines (Tabosa et al., 2004), and sporadically in captive bears, a camel, and a central America jaguar (Camus et al., 2004; Grooters, 2003). Cases of pythiosis in humans are rare in most endemic areas. However, Thailand possesses the highest rate of human pythiosis with some 90 cases diagnosed since 1987 (Wanachiwanawin et al., 2004). Bovine pythiosis was first reported by Miller et al. (1985) in the USA. These investigators considered pythiosis an exotic bovine disease. The report of two cases of bovine pythiosis in Brazil, 13 years later (Santurio et al., 1998), tends to support this observation. Enzootic bovine granulomatosis (EBG) affects beef calves in the flooded Savanna regions of Venezuela (Marin et al., 1991). The primary clinical manifestations involved lesions of the limbs, which influences the development and quality of life of infected calves. The studies of Marin et al. (1991) suggested that a fungal etiology or P. insidiosum were the most likely agents involved in this pathology. However, no other studies were conducted to validate their assumption. In this study, we report enzootic pythiosis in beef calves inhabiting the flooding Venezuelan Savannas in the State of Apure. Sixty-three calves with pythiosis were found during a 2-year study. This report deals with the description of three outbreaks of bovine pythiosis in Venezuela.
2. Materials and methods 2.1. Selected animals and clinical sample collection Between 2002 and 2004, three outbreaks of EBG, affecting 63 Brahman beef calves in the State of Apure, Venezuela, were studied. All cases showed subcutaneous and cutaneous involvement of the lower extremities, primarily in the distal portion of their limbs. Tissue samples were collected from the 63 affected calves, cut into small pieces and send for microbiological analyses or fixed in formaldehyde, sectioned, stained with hematoxylin and eosine (H&E), methenamine silver de Gomori and with a combination of H&E and silver stain, for histopathological analysis. 2.2. Anti-Pythium insidiosum histochemical assay To further investigate the identity of the hyphal elements present in the studied infected tissues, in situ direct flourescent antibodies (FA) against P. insidiosum were used on the 63 infected beef calves. The antiserum and fluorescent labeling was as previously described (Mendoza et al., 1987; Reis et al., 2003). Briefly, unstained tissue sections, free of paraffin, were stained with one or two drops of anti-P. insidiosum reagent and then incubated for 30 min at room temperature and then observed with fluorescent microscope. Control tissue used during this analysis included hyphal elements of Conidiobolus coronatus, Basidiobolus ranarum, and Aspergillus spp. 2.3. Serological analyses Four serological assays, enzyme-linked immunosorbent assay (ELISA) (Mendoza et al., 1997), immunodiffusion (ID) (Mendoza et al., 1986), latex agglutination (LA) (Hutchens and Mendoza, 2002), and western blot (WB) (Mendoza and Nicholson, 1992), were used to evaluate the presence of anti-P. insidiosum immunoglobulins in the sera of the infected calves (Table 1). The antigen used on all assays was prepared following the authors recommendations using the type strain ATCC 58643 = CBS 574-85 in 2% Sabouraud dextrose broth. Only the sera from 57 cases were investigated with all four
R.C. Pe´rez et al. / Veterinary Microbiology 109 (2005) 121–128
123
Table 1 Summary of the results obtained using four different immunological assays on 57 bovine sera Assay
Number of tested sera
Positive
Negative
Immunodiffusion
57
13 (22.8%)
44 (77.2%)
Latex agglutination
57
++++/+++, ++/+ 24 (42.1%), 29 (50.9%)
4 (7%)
ELISA Western blot
57 57
57 (100%) 57 (100%)
0 0
Immunodiffusion was the most insensitive of all assays. The sensitivity of the latex agglutination was satisfactory but could not be compared with the high sensitivity of the ELISA and Western blot.
serological assays (Table 1). The diagnosis of pythiosis in the remaining six calves was confirmed with the used of FA in the collected tissue samples. This group of animals belong to the last outbreak of the disease studied in 2004. 2.4. Microbiological analysis Biopsed tissues samples were cut into small 2.0 mm diameter pieces. Some pieces were used for wet mount preparations and the remaining for culture. Samples for wet mount analysis were placed on a slide with one or two drops of 10% KOH plus ink-blue stain, incubated at room temperature for 15 min, and then examined under light microscopy. Pieces of the infected tissues were cultured onto 2% dextrose Sabouraud agar and then incubated at 37 and 25 8C for 5–10 days.
3. Results 3.1. Epidemiological features and clinical symptoms There are two seasons in Venezuela, the rainy season, between April and October characterized by heavy flooding, and a dry season, from November to March with the accumulation of stagnant water and high temperatures (27–39 8C). The selected cases came from six different nearby herds located on ‘‘El Alto de Apure’’, a flooded Savanna region of this country with high temperatures and excessive precipitation. Beef calves are usually in contact with these areas during the rainy season because there are few dry areas left, and during the dry season because of the need for water. Although the majority of cases
of EBG occurred during the rainy season, this pathology was observed all year around. Before contracting the disease, the studied animals were observed submerged in the wet areas. Once infected, the classical clinical symptoms were pruritus, edema, and claudication with secondary bacterial contamination. All lesions were located on their limbs, especially on the distal portions of these anatomical areas. A similar number of males and females were found with the disease. The affected areas were extremely painful and most affected calves could not stand up, a situation that leads to weigh lost and eventually to starvation and death. Lesions located on the chest, thorax, abdomen, and mammary gland were not observed. The lesions had developed approximately between 1 and 6 months (85%) especially in young calves (1-yearold or less), and 1–2 years (15%) in older calves. The actual occurrence of the disease is unknown, but data collected in this study suggest a 4.2% annual rate in the studied areas. 3.2. Pathological findings 3.2.1. Gross pathology The calves studied showed granulomatous tumorallike lesions localized on the distal portion of the metacarpal, metatarsal and/or phalangeal anatomical areas. The swelling lesions appeared either ulcerated or without ulcers. The swelling areas were characterized by presence of non-ulcerated lesions with multifocal sinus tracts draining a serosanguineous material (Fig. 1). Some swelling of non-ulcerated lesions showed the presence of pus when surgically intervened. Ulcerated lesions were usually circular in shape and variable in size (50 to 100 mm 100 mm) (Fig. 2). This type of pathology was character-
124
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
Fig. 1. The figure depicts two calves (A and B) with extensive swelling non-ulcerated lesions on the distal metacarpal and phalangeal anatomical areas. The arrows show the presence of sinus tracts observed in these type skin lesions.
ized by the presence of several pockets of necrotic tissue. The hard stony masses termed ‘‘kunkers’’, a typical feature of equine pythiosis, were not found. 3.2.2. Histopathology and anti-Pythium insidiosum histochemical assay On H&E, the collected samples showed multifocal necrotic areas with numerous giant cells, eosinophils, neutrophils, macrophages, and plasma cells (Fig. 3). On high magnification, the presence of short poorly stained hyaline hyphal filaments were observed within some giant cells and in the necrotic areas (Fig. 3). The
presence of a prominent eosinophilic reaction around the hyphal elements (Splendore–Hoeppli phenomenon) was a characteristic feature of the invading hyphae. Sections of the collected skin samples stained with silver, showed the presence of 4–6 mm in diameter sparsely septate hyphal elements compatible with those observed in cases of pythiosis (Fig. 3). The FA assay using anti-P. insidiosum specific serum, identified the hyphal elements in the infected tissues of the 63 infected studied calves as P. insidiosum. The control tissues used during this study were always negative.
Fig. 2. The calves in the figure (A, B, and C) showed ulcerated lesions on the distal metacarpal or metatarsal areas caused by Pythium insidiosum. A main feature of the ulcerate lesions was their circular shape and the absence of the stony masses termed ‘‘kunkers’’, a characteristic of equine pythiosis.
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
125
Fig. 3. Panel A shows granulomatous tissue with numerous giant cells, polymorphonuclear cells, and eosinophils (H&E, 20). Panel B shows a section of the skin with a subcutaneous granulomatous lesion containing hyphae (white rectangle) (H&E, 20). Poorly stained hyphal elements of Pythium insidiosum are observed within the giant cells (lower section of Panel A; H&E, 100) and in other areas of the infected tissues (upper section of Panel A; silver, 100). The upper section of Panel B showed details of the rectangular area containing hyphae elements of Pythium insidiosum (white arrow, 50).
3.3. Serological assays The results of LA are shown in Table 1. In summary, 42.1% of the samples showed strong agglutination (+++/++++), 50.9% of the evaluated sera showed a weak reaction (+/++), whereas 7% (four calves) did not react. Immunodiffusion test showed only 13 positive sera with two to three identity bands. All positive samples in ID were also positive in the latex agglutination test (Table 1). When the collected sera were evaluated on ELISA, 100% of the sera showed positive reaction with optical density (OD) readings ranging 0.468–1.470 (cut off point 0.262 OD). Six sera from apparently healthy calves from the same region were also positive in the ELISA. Similar results were observed in WB. In this sensitive assay, the sera from infected calves detected several P. insidiosum immunodominant high molecular weight antigens, ranging from 203 to 51 kiloDalton (kDa) and a single (32 kDa) or doublet 32–30 kDa low molecular antigens (Fig. 4). Some antibodies in the tested sera detected a doublet at 32 kDa, whereas other bovines only have a single band at this particular molecular mass. 3.4. Microbiological analysis Tissue samples observed in 10% KOH showed the presence of slender, sparsely septate hyaline branched
Fig. 4. Western immunoblot analysis carried out on the sera of three infected calves (Lanes 1–3). The presence of antigenic proteins ranging from 203, 120–51 kDa is depicted. A 32 kDa band and a 32– 30 kDa doublet are also showed in this figure. Some sera possess a doublet at the 32–30 kDa molecular weight, whereas others have a single band. Numbers on the left indicate molecular mass markers (in kiloDaltons, BioRad).
126
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
hyphae, sometimes surrounded by a fine granular diffused material. The morphological features of the observed structures were compatible with the hyphal filaments of P. insidiosum. No hyphal forms were observed on the pus collected from secondarily contaminated lesions. Tissue samples inoculated onto 2% Sabouraud dextrose agar and incubated at 37 and 25 8C with increased humidity, showed heavy contamination with thermophilic fungal and bacterial species. All attempts to recover P. insidiosum from the infected tissues were fruitless.
4. Discussion For years, several cattle breeding farms in the State of Apure, Venezuela, have been increasingly reporting a pathological entity in beef calves inflicting economical distress for the affected ranchers. Although pythiosis in beef calves was previously diagnosed in the USA (Miller et al., 1985) and Brazil (Santurio et al., 1998), these reports were limited to single cases of bovine pythiosis. However, this study found, for the first time, epizootic pythiosis with the involvement of several animals. Based on this finding we believe that enzootic bovine pythiosis (EBP) should be considered in cases of skin bovine lesions of the limbs, especially on those farms located in the enzootic areas of the disease. The clinical and pathological changes described by Miller et al. (1985) and Santurio et al. (1998) were similar to those observed in cases of EBP depicted in this study. For instance, lesions were limited to the lower portion of their limbs, have multifocal sinus tracts, ulcerated tissues and lack the presence of hard stony masses known as ‘‘kunkers’’. One interesting aspect in this study, previously not reported, was the presence of extensive ulcerated tissues in some infected calves, very similar to the skin lesions of pythiosis reported in dogs (Thomas and Lewis, 1998), horses (Alfaro and Mendoza, 1990; Chaffin et al., 1995; Miller, 1981), sheep (Tabosa et al., 2004), and humans (Thianprasit, 1990) with pythiosis. The histopathological analyses in the studied calves were characterized by the presence of eosinophilic granulomatous lesions with numerous giant cells engulfing hyphal elements of P. insidiosum, a characteristic in common with pathological changes observed previously in calves from the USA (Miller et al., 1985)
and Brazil (Santurio et al., 1998). Although pythiosis in humans and other animals is reported affecting one or two individuals rather than in outbreaks, in this study we found three or more infected animals per farm. A similar observation was recently described in Brazilian sheep with pythiosis (Tabosa et al., 2004). These findings suggest that skin pythiosis in bovines and ovines could occur as outbreaks in the enzootic areas of pythiosis, and therefore, this disease may be of economic relevance in these species. Serological techniques have been instrumental in the study of pythiosis (Hutchens and Mendoza, 2002; Grooters, 2003; Mendoza et al., 1986, 1997; Mendoza and Nicholson, 1992; Vanittanakom et al., 2004). We took advantage of these techniques to demonstrate the presence of anti-P. insidiosum immunoglobulins in 57 of the infected calves studied. As previously reported (Mendoza et al., 1986), the ID test was the most insensitive assay followed by the LA, a newly introduced screening test for pythiosis, characterized by its high sensitivity, but with a high rate of false positives (Hutchens and Mendoza, 2002). The enzymatic assays (ELISA and WB) detected 100% of the infected calves. Both assays, however, were positive when sera from apparently normal calves collected in the enzootic areas were tested. This finding suggests that animals in the enzootic areas have been in contact with P. insidiosum without developing the disease, since the sera from apparently normal bovines from non-enzootic areas were negative in the same assays. Interestingly, the protein profile observed in WB, using sera from bovine with the disease, did not show the same profile previously reported in horses with cutaneous pythiosis (Mendoza and Nicholson, 1992). This might indicate that different species could trigger specific immunoresponse to different antigens, or that the antigen used in our analysis was prepared in a different medium than the one used by Mendoza and Nicholson (1992). Culture is the golden test for most infectious disease. However, it is time consuming and insensitive. In this study, despite numerous attempts, the isolation of P. insidosum was not possible. This is in contrast with the two first-reports from the USA and Brazil in which the pathogen was isolated from 80% of the implants (Miller et al., 1985; Santurio et al., 1998). One explanation for this failure could be that in this study samples were collected 2–3 days before culture.
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
Although samples were kept at all times in water with several antibiotics, and were vigorously washed before inoculation on 2% dextrose Sabouraud agar, the presence of fast growing thermophilic fungi and resistant bacteria covered the plates in less than 24 h. Immunohistochemical analysis proved to be accurate methods for the identification of P. insidiosum in infected humans and animals with pythiosis (Mendoza et al., 1987; Reis et al., 2003). Because of culture failure, in situ fluorescence antibodies were used. This assay showed that the identity of the hyphal elements present in the infected calves belongs to P. insidiosum, confirming the importance of this test in the absence of culture. All in all, this study showed that EBP is an important pathology in the flooding Savannas of Venezuela and that it probably also occurs in other enzootic areas of the world, but its economic relevance in those areas remains unknown. This study suggests that the presence of skin lesions with sinus tracts and ulcers on the limbs of beef calves inhabiting breeding farms of tropical and subtropical areas should alert clinicians on this novel pathology of bovines.
Acknowledgments The authors thanks Dr. Ariya Chindamporn for her help on some of the serological assays performed on the sera from the infected calves. We also want to thanks Drs. Nancy Viera, Laura Real de Pinero, Luisa Gonzalez, and Francisco Triana-Alonso for their assistance during this study. This study was supported in part by the Medical Technology Program, Michigan State University.
References Alfaro, A.A., Mendoza, L., 1990. Four cases of equine bone lesions caused by Pythium insidosum. Equine Vet. J. 22, 295–297. Camus, A.C., Grooters, A.M., Aquilar, R.E., 2004. Granulomatous pneumonia caused by Pythium insidiosum in a central American jaguar Panthera onca. J. Vet. Diagn. Invest. 16, 567–571. Chaffin, M.K., Schumacher, J., McMullan, W.C., 1995. Cutaneous pythiosis in the horse. Vet. Clin. N. Am. Equine Pract. 11, 91– 103. Grooters, A.M., 2003. Pythiosis, lagenidiosis, and zygomycosis in small animals. Vet. Clin. Small Anim. 33, 695–720.
127
Hutchens, M., Mendoza, L., May 2002. Development of a simplified latex agglutination test for the rapid diagnosis of the infections caused by Pythium insidiosum. In: 102nd General Meeting of the American Society for Microbiology, Salt Lake City, Utah, USA, p. 124. Hutchins, D.R., Johnston, K., 1972. Phycomycosis in the horse. Aust. Vet. J. 48, 269–278. Hensel, P., Greene, C.E., Medleau, L., Latimer, K.S., Mendoza, l., 2003. Immunotherapy for treatment of multicentric cutaneous pythiosis in a dog. J. Am. Vet. Med. Assoc. 223, 215–218. Marin, C., Lopez, N., Perez, C., Mirabal, G., Rolo, M., Urdaneta, J., 1991. Primer reporte de la Granulomatosis Enzootica Bovina en Venezuela. Fonaiap Divulga 37, 15–16. McMullan, W.C., Joyce, J.R., Janselka, D.V., Heitman, J.M., 1977. Amphotericin B for the treatment of localized subcutaneous phycomycosis in the horse. J. Am. Vet. Med. Assoc. 170, 1293– 1298. Mendoza, L., Kaufman, L., Standard, P., 1986. Immunodiffusion test for diagnosis and monitoring pythiosis in horses. J. Clin. Microbiol. 23, 813–816. Mendoza, L., Kaufman, L., Standard, P., 1987. Antigenic relationship between between the animal and human pathogen Pythium insidiosum. J. Clin. Microbiol. 25, 2159–2162. Mendoza, L., Nicholson, V., Precott, J.F, 1992. Immonoblot analysis of the humoral immune response to Pythium insidiosum in horses with pythiosis. J. Clin. Microbiol. 30, 2980–2983. Mendoza, L., Ajello, L., McGinnis, L., 1996. Infections caused by the oomycetous pathogen Pythium insidiosum. J. Mycol. Med. 6, 151–164. Mendoza, L., Kaufman, L., Mandy, W., Glass, R., 1997. Serodiagnosis of human and animal pythiosis using an enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol. 4, 715–718. Mendoza, L., Mandy, W., Glass, B., 2003. An improved Pythium insidiosum-vaccine formulation with enhanced immunotherapeutic properties in horses and dogs with pythiosis. Vaccine 21, 2797–2804. Miller, I.R., 1981. Treatment of equine phycomycosis by immunotherapy and surgery. Aust. Vet. J. 57, 377–382. Miller, R.I., Olcott, B.M., Archer, M., 1985. Cutaneous pythiosis in beef calves. J. Am. Vet. Med. Assoc. 186, 984–986. Reis, J.L., Quiroz de Carvalho, E.C., Nogueira, R.H.G., Lemos, L.S., Mendoza, L., 2003. Disseminated pythiosis in three horses. Vet. Microbiol. 96, 289–295. Santurio, J.M., Monteiro, A.B., Leal, A.T., Kommers, G.D., de Sousa, R.S., Catto, J.B., 1998. Cutaneous pythiosis in calves from the Pantanal region of Brazil. Mycopathologia 141, 123–125. Thianprasit, M., 1990. Human pythiosis. Trop. Dermatol. 4, 1–4. Tabosa, I.M., Riet-Correa, F., Nobre, V.M.T., Azevedo, E.O., Reis Jr., J.L., Medeiros, R.M.T., 2004. Outbreaks of pythiosis in two flocks of sheep in Northeastern Brazil. Vet. Pathol. 41, 412–415. Thiatithanyanont, A., Mendoza, L., Chuansumrit, A., Pracharktam, R., Laothamatas, J., Sathapatayavongs, B., Lolekha, S., Ajello, L., 1998. Use of an immunotherapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum. Clin. Infect. Dis. 27, 1394–1400. Thomas, R.C., Lewis, D.T., 1998. Pythiosis in dogs and cats. Compendium 20, 63–75.
128
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
Vanittanakom, N., Supabandhu, J., Khamwan, C., Praparattanapan, J., Thirach, S., Prasertwitayakij, N., Louthrenoo, W., Chiewchanvit, S., Napaporn Tananuvat, N., 2004. Identification of emerging human-pathogenic Pythium insidiosum by serological and molecular assay-based methods. J. Clin. Microbiol. 42, 3970–3974.
Wanachiwanawin, W., Mendoza, L., Visuthisakchai, S., Mutsikapan, P., Sathapatayavongs, B., Chaiprasert, A., Suwanagool, P., Manuskiatti, W., Ruangsetakit, C., Ajello, L., 2004. Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans. Vaccine 22, 3613– 3621.
Epizootic cutaneous pythiosis in beef calves Rosa Cristina Pe´rez a, Juan J. Luis-Leo´n a,b, Jose´ Luis Vivas c, Leonel Mendoza d,* a
University of Carabobo, Faculty of Health Science Health, Department of Microbiology (Aragua), Venezuela b Center of Biomedical Research, University of Carabobo (BIOMED-UC), Venezuela c Laboratory Clinic Lugo ‘‘Delgado Launois’’, Maracay, Estado de Aragua, Venezuela d Medical Technology Program, Microbiology and Molecular Genetics, Michigan State University, 322 North Kedzie Hall, East Lansing, MI 48824-1031, USA Received 30 December 2004; received in revised form 16 April 2005; accepted 19 April 2005
Abstract Enzootic bovine granulomatosis (EBG) is a disease that affects beef calves in the flooded Savanna regions of Venezuela. Although Pythium insidiosum was originally the suspected etiologic agent, nothing was done to demonstrate its presence in the infected animals. The objective of this study was to investigate the etiologic agent causing cutaneous lesions in a group of 63 calves diagnosed with EBG. The collected samples were analyzed by histopathology, immunohistochemistry, serology, and mycological techniques. The clinical manifestations of BEG included ulcerated and swollen granulomatous lesions on the limbs of young calves. Histopathological studies revealed an eosinophilic inflammatory reaction with neutrophils, giant cells and branching hyphae with the Splendore–Hoeppli phenomenon. Immunofluorescence analysis identified the branching hyphae as elements of the fungal-like organism P. insidiosum. Wet mounts in 10% KOH revealed hyaline slender branching hyaline coenocytic hyphae, but cultures from those samples were negative. Anti-P. insidiosum immunoglobulins were also demonstrated in the collected sera of the affected bovines using latex agglutination, immunodiffusion, ELISA, and Western Blot. The histopathological, immunohistochemical, and serological tests showed for the first time that P. insidiosum could be involved in epizootic bovine pythiosis. # 2005 Elsevier B.V. All rights reserved. Keywords: Bovine pythiosis; Epizootic pythiosis; Pythium insidiosum
1. Introduction Pythiosis is a life threatening disease of the tropical, subtropical, and temperate areas of the * Corresponding author. Tel.: +1 517 353 7800; fax: +1 517 432 2006. E-mail address: [email protected] (L. Mendoza).
world. It is characterized by the development chronic lesions on the cutaneous and subcutaneous, intestinal, and bone tissues, as well as invasion of blood vessels in the infected animals and humans (Mendoza et al., 1996). The etiologic agent is the aquatic fungal-like striminipilan pathogen Pythium insidiosum. The infections are acquired when susceptible animals
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2005.04.020
122
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
and humans contact the infecting propagules of this pathogen through traumatic lesions in the enzootic areas. If the disease is not treated in the early stages, the infected hosts often die within weeks (Chaffin et al., 1995; Thomas and Lewis, 1998; Thianprasit, 1990). Chemotherapy using antifungal drugs has been difficult (Hutchins and Johnston, 1972; McMullan et al., 1977) and traditional treatments such as surgery and iodides produce limited results (Hutchins and Johnston, 1972; Miller, 1981; Thianprasit, 1990). More recently, immunotherapy has showed a promising new way to treat equines, dogs, and humans with the disease (Hensel et al., 2003; Mendoza et al., 2003; Thiatithanyanont et al., 1998; Wanachiwanawin et al., 2004). Pythiosis is more frequently diagnosed in equines and dogs (Alfaro and Mendoza, 1990; Chaffin et al., 1995; Thomas and Lewis, 1998) with few cases reported in bovines (Miller et al., 1985; Santurio et al., 1998), felines (Thomas and Lewis, 1998), ovines (Tabosa et al., 2004), and sporadically in captive bears, a camel, and a central America jaguar (Camus et al., 2004; Grooters, 2003). Cases of pythiosis in humans are rare in most endemic areas. However, Thailand possesses the highest rate of human pythiosis with some 90 cases diagnosed since 1987 (Wanachiwanawin et al., 2004). Bovine pythiosis was first reported by Miller et al. (1985) in the USA. These investigators considered pythiosis an exotic bovine disease. The report of two cases of bovine pythiosis in Brazil, 13 years later (Santurio et al., 1998), tends to support this observation. Enzootic bovine granulomatosis (EBG) affects beef calves in the flooded Savanna regions of Venezuela (Marin et al., 1991). The primary clinical manifestations involved lesions of the limbs, which influences the development and quality of life of infected calves. The studies of Marin et al. (1991) suggested that a fungal etiology or P. insidiosum were the most likely agents involved in this pathology. However, no other studies were conducted to validate their assumption. In this study, we report enzootic pythiosis in beef calves inhabiting the flooding Venezuelan Savannas in the State of Apure. Sixty-three calves with pythiosis were found during a 2-year study. This report deals with the description of three outbreaks of bovine pythiosis in Venezuela.
2. Materials and methods 2.1. Selected animals and clinical sample collection Between 2002 and 2004, three outbreaks of EBG, affecting 63 Brahman beef calves in the State of Apure, Venezuela, were studied. All cases showed subcutaneous and cutaneous involvement of the lower extremities, primarily in the distal portion of their limbs. Tissue samples were collected from the 63 affected calves, cut into small pieces and send for microbiological analyses or fixed in formaldehyde, sectioned, stained with hematoxylin and eosine (H&E), methenamine silver de Gomori and with a combination of H&E and silver stain, for histopathological analysis. 2.2. Anti-Pythium insidiosum histochemical assay To further investigate the identity of the hyphal elements present in the studied infected tissues, in situ direct flourescent antibodies (FA) against P. insidiosum were used on the 63 infected beef calves. The antiserum and fluorescent labeling was as previously described (Mendoza et al., 1987; Reis et al., 2003). Briefly, unstained tissue sections, free of paraffin, were stained with one or two drops of anti-P. insidiosum reagent and then incubated for 30 min at room temperature and then observed with fluorescent microscope. Control tissue used during this analysis included hyphal elements of Conidiobolus coronatus, Basidiobolus ranarum, and Aspergillus spp. 2.3. Serological analyses Four serological assays, enzyme-linked immunosorbent assay (ELISA) (Mendoza et al., 1997), immunodiffusion (ID) (Mendoza et al., 1986), latex agglutination (LA) (Hutchens and Mendoza, 2002), and western blot (WB) (Mendoza and Nicholson, 1992), were used to evaluate the presence of anti-P. insidiosum immunoglobulins in the sera of the infected calves (Table 1). The antigen used on all assays was prepared following the authors recommendations using the type strain ATCC 58643 = CBS 574-85 in 2% Sabouraud dextrose broth. Only the sera from 57 cases were investigated with all four
R.C. Pe´rez et al. / Veterinary Microbiology 109 (2005) 121–128
123
Table 1 Summary of the results obtained using four different immunological assays on 57 bovine sera Assay
Number of tested sera
Positive
Negative
Immunodiffusion
57
13 (22.8%)
44 (77.2%)
Latex agglutination
57
++++/+++, ++/+ 24 (42.1%), 29 (50.9%)
4 (7%)
ELISA Western blot
57 57
57 (100%) 57 (100%)
0 0
Immunodiffusion was the most insensitive of all assays. The sensitivity of the latex agglutination was satisfactory but could not be compared with the high sensitivity of the ELISA and Western blot.
serological assays (Table 1). The diagnosis of pythiosis in the remaining six calves was confirmed with the used of FA in the collected tissue samples. This group of animals belong to the last outbreak of the disease studied in 2004. 2.4. Microbiological analysis Biopsed tissues samples were cut into small 2.0 mm diameter pieces. Some pieces were used for wet mount preparations and the remaining for culture. Samples for wet mount analysis were placed on a slide with one or two drops of 10% KOH plus ink-blue stain, incubated at room temperature for 15 min, and then examined under light microscopy. Pieces of the infected tissues were cultured onto 2% dextrose Sabouraud agar and then incubated at 37 and 25 8C for 5–10 days.
3. Results 3.1. Epidemiological features and clinical symptoms There are two seasons in Venezuela, the rainy season, between April and October characterized by heavy flooding, and a dry season, from November to March with the accumulation of stagnant water and high temperatures (27–39 8C). The selected cases came from six different nearby herds located on ‘‘El Alto de Apure’’, a flooded Savanna region of this country with high temperatures and excessive precipitation. Beef calves are usually in contact with these areas during the rainy season because there are few dry areas left, and during the dry season because of the need for water. Although the majority of cases
of EBG occurred during the rainy season, this pathology was observed all year around. Before contracting the disease, the studied animals were observed submerged in the wet areas. Once infected, the classical clinical symptoms were pruritus, edema, and claudication with secondary bacterial contamination. All lesions were located on their limbs, especially on the distal portions of these anatomical areas. A similar number of males and females were found with the disease. The affected areas were extremely painful and most affected calves could not stand up, a situation that leads to weigh lost and eventually to starvation and death. Lesions located on the chest, thorax, abdomen, and mammary gland were not observed. The lesions had developed approximately between 1 and 6 months (85%) especially in young calves (1-yearold or less), and 1–2 years (15%) in older calves. The actual occurrence of the disease is unknown, but data collected in this study suggest a 4.2% annual rate in the studied areas. 3.2. Pathological findings 3.2.1. Gross pathology The calves studied showed granulomatous tumorallike lesions localized on the distal portion of the metacarpal, metatarsal and/or phalangeal anatomical areas. The swelling lesions appeared either ulcerated or without ulcers. The swelling areas were characterized by presence of non-ulcerated lesions with multifocal sinus tracts draining a serosanguineous material (Fig. 1). Some swelling of non-ulcerated lesions showed the presence of pus when surgically intervened. Ulcerated lesions were usually circular in shape and variable in size (50 to 100 mm 100 mm) (Fig. 2). This type of pathology was character-
124
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
Fig. 1. The figure depicts two calves (A and B) with extensive swelling non-ulcerated lesions on the distal metacarpal and phalangeal anatomical areas. The arrows show the presence of sinus tracts observed in these type skin lesions.
ized by the presence of several pockets of necrotic tissue. The hard stony masses termed ‘‘kunkers’’, a typical feature of equine pythiosis, were not found. 3.2.2. Histopathology and anti-Pythium insidiosum histochemical assay On H&E, the collected samples showed multifocal necrotic areas with numerous giant cells, eosinophils, neutrophils, macrophages, and plasma cells (Fig. 3). On high magnification, the presence of short poorly stained hyaline hyphal filaments were observed within some giant cells and in the necrotic areas (Fig. 3). The
presence of a prominent eosinophilic reaction around the hyphal elements (Splendore–Hoeppli phenomenon) was a characteristic feature of the invading hyphae. Sections of the collected skin samples stained with silver, showed the presence of 4–6 mm in diameter sparsely septate hyphal elements compatible with those observed in cases of pythiosis (Fig. 3). The FA assay using anti-P. insidiosum specific serum, identified the hyphal elements in the infected tissues of the 63 infected studied calves as P. insidiosum. The control tissues used during this study were always negative.
Fig. 2. The calves in the figure (A, B, and C) showed ulcerated lesions on the distal metacarpal or metatarsal areas caused by Pythium insidiosum. A main feature of the ulcerate lesions was their circular shape and the absence of the stony masses termed ‘‘kunkers’’, a characteristic of equine pythiosis.
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
125
Fig. 3. Panel A shows granulomatous tissue with numerous giant cells, polymorphonuclear cells, and eosinophils (H&E, 20). Panel B shows a section of the skin with a subcutaneous granulomatous lesion containing hyphae (white rectangle) (H&E, 20). Poorly stained hyphal elements of Pythium insidiosum are observed within the giant cells (lower section of Panel A; H&E, 100) and in other areas of the infected tissues (upper section of Panel A; silver, 100). The upper section of Panel B showed details of the rectangular area containing hyphae elements of Pythium insidiosum (white arrow, 50).
3.3. Serological assays The results of LA are shown in Table 1. In summary, 42.1% of the samples showed strong agglutination (+++/++++), 50.9% of the evaluated sera showed a weak reaction (+/++), whereas 7% (four calves) did not react. Immunodiffusion test showed only 13 positive sera with two to three identity bands. All positive samples in ID were also positive in the latex agglutination test (Table 1). When the collected sera were evaluated on ELISA, 100% of the sera showed positive reaction with optical density (OD) readings ranging 0.468–1.470 (cut off point 0.262 OD). Six sera from apparently healthy calves from the same region were also positive in the ELISA. Similar results were observed in WB. In this sensitive assay, the sera from infected calves detected several P. insidiosum immunodominant high molecular weight antigens, ranging from 203 to 51 kiloDalton (kDa) and a single (32 kDa) or doublet 32–30 kDa low molecular antigens (Fig. 4). Some antibodies in the tested sera detected a doublet at 32 kDa, whereas other bovines only have a single band at this particular molecular mass. 3.4. Microbiological analysis Tissue samples observed in 10% KOH showed the presence of slender, sparsely septate hyaline branched
Fig. 4. Western immunoblot analysis carried out on the sera of three infected calves (Lanes 1–3). The presence of antigenic proteins ranging from 203, 120–51 kDa is depicted. A 32 kDa band and a 32– 30 kDa doublet are also showed in this figure. Some sera possess a doublet at the 32–30 kDa molecular weight, whereas others have a single band. Numbers on the left indicate molecular mass markers (in kiloDaltons, BioRad).
126
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
hyphae, sometimes surrounded by a fine granular diffused material. The morphological features of the observed structures were compatible with the hyphal filaments of P. insidiosum. No hyphal forms were observed on the pus collected from secondarily contaminated lesions. Tissue samples inoculated onto 2% Sabouraud dextrose agar and incubated at 37 and 25 8C with increased humidity, showed heavy contamination with thermophilic fungal and bacterial species. All attempts to recover P. insidiosum from the infected tissues were fruitless.
4. Discussion For years, several cattle breeding farms in the State of Apure, Venezuela, have been increasingly reporting a pathological entity in beef calves inflicting economical distress for the affected ranchers. Although pythiosis in beef calves was previously diagnosed in the USA (Miller et al., 1985) and Brazil (Santurio et al., 1998), these reports were limited to single cases of bovine pythiosis. However, this study found, for the first time, epizootic pythiosis with the involvement of several animals. Based on this finding we believe that enzootic bovine pythiosis (EBP) should be considered in cases of skin bovine lesions of the limbs, especially on those farms located in the enzootic areas of the disease. The clinical and pathological changes described by Miller et al. (1985) and Santurio et al. (1998) were similar to those observed in cases of EBP depicted in this study. For instance, lesions were limited to the lower portion of their limbs, have multifocal sinus tracts, ulcerated tissues and lack the presence of hard stony masses known as ‘‘kunkers’’. One interesting aspect in this study, previously not reported, was the presence of extensive ulcerated tissues in some infected calves, very similar to the skin lesions of pythiosis reported in dogs (Thomas and Lewis, 1998), horses (Alfaro and Mendoza, 1990; Chaffin et al., 1995; Miller, 1981), sheep (Tabosa et al., 2004), and humans (Thianprasit, 1990) with pythiosis. The histopathological analyses in the studied calves were characterized by the presence of eosinophilic granulomatous lesions with numerous giant cells engulfing hyphal elements of P. insidiosum, a characteristic in common with pathological changes observed previously in calves from the USA (Miller et al., 1985)
and Brazil (Santurio et al., 1998). Although pythiosis in humans and other animals is reported affecting one or two individuals rather than in outbreaks, in this study we found three or more infected animals per farm. A similar observation was recently described in Brazilian sheep with pythiosis (Tabosa et al., 2004). These findings suggest that skin pythiosis in bovines and ovines could occur as outbreaks in the enzootic areas of pythiosis, and therefore, this disease may be of economic relevance in these species. Serological techniques have been instrumental in the study of pythiosis (Hutchens and Mendoza, 2002; Grooters, 2003; Mendoza et al., 1986, 1997; Mendoza and Nicholson, 1992; Vanittanakom et al., 2004). We took advantage of these techniques to demonstrate the presence of anti-P. insidiosum immunoglobulins in 57 of the infected calves studied. As previously reported (Mendoza et al., 1986), the ID test was the most insensitive assay followed by the LA, a newly introduced screening test for pythiosis, characterized by its high sensitivity, but with a high rate of false positives (Hutchens and Mendoza, 2002). The enzymatic assays (ELISA and WB) detected 100% of the infected calves. Both assays, however, were positive when sera from apparently normal calves collected in the enzootic areas were tested. This finding suggests that animals in the enzootic areas have been in contact with P. insidiosum without developing the disease, since the sera from apparently normal bovines from non-enzootic areas were negative in the same assays. Interestingly, the protein profile observed in WB, using sera from bovine with the disease, did not show the same profile previously reported in horses with cutaneous pythiosis (Mendoza and Nicholson, 1992). This might indicate that different species could trigger specific immunoresponse to different antigens, or that the antigen used in our analysis was prepared in a different medium than the one used by Mendoza and Nicholson (1992). Culture is the golden test for most infectious disease. However, it is time consuming and insensitive. In this study, despite numerous attempts, the isolation of P. insidosum was not possible. This is in contrast with the two first-reports from the USA and Brazil in which the pathogen was isolated from 80% of the implants (Miller et al., 1985; Santurio et al., 1998). One explanation for this failure could be that in this study samples were collected 2–3 days before culture.
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
Although samples were kept at all times in water with several antibiotics, and were vigorously washed before inoculation on 2% dextrose Sabouraud agar, the presence of fast growing thermophilic fungi and resistant bacteria covered the plates in less than 24 h. Immunohistochemical analysis proved to be accurate methods for the identification of P. insidiosum in infected humans and animals with pythiosis (Mendoza et al., 1987; Reis et al., 2003). Because of culture failure, in situ fluorescence antibodies were used. This assay showed that the identity of the hyphal elements present in the infected calves belongs to P. insidiosum, confirming the importance of this test in the absence of culture. All in all, this study showed that EBP is an important pathology in the flooding Savannas of Venezuela and that it probably also occurs in other enzootic areas of the world, but its economic relevance in those areas remains unknown. This study suggests that the presence of skin lesions with sinus tracts and ulcers on the limbs of beef calves inhabiting breeding farms of tropical and subtropical areas should alert clinicians on this novel pathology of bovines.
Acknowledgments The authors thanks Dr. Ariya Chindamporn for her help on some of the serological assays performed on the sera from the infected calves. We also want to thanks Drs. Nancy Viera, Laura Real de Pinero, Luisa Gonzalez, and Francisco Triana-Alonso for their assistance during this study. This study was supported in part by the Medical Technology Program, Michigan State University.
References Alfaro, A.A., Mendoza, L., 1990. Four cases of equine bone lesions caused by Pythium insidosum. Equine Vet. J. 22, 295–297. Camus, A.C., Grooters, A.M., Aquilar, R.E., 2004. Granulomatous pneumonia caused by Pythium insidiosum in a central American jaguar Panthera onca. J. Vet. Diagn. Invest. 16, 567–571. Chaffin, M.K., Schumacher, J., McMullan, W.C., 1995. Cutaneous pythiosis in the horse. Vet. Clin. N. Am. Equine Pract. 11, 91– 103. Grooters, A.M., 2003. Pythiosis, lagenidiosis, and zygomycosis in small animals. Vet. Clin. Small Anim. 33, 695–720.
127
Hutchens, M., Mendoza, L., May 2002. Development of a simplified latex agglutination test for the rapid diagnosis of the infections caused by Pythium insidiosum. In: 102nd General Meeting of the American Society for Microbiology, Salt Lake City, Utah, USA, p. 124. Hutchins, D.R., Johnston, K., 1972. Phycomycosis in the horse. Aust. Vet. J. 48, 269–278. Hensel, P., Greene, C.E., Medleau, L., Latimer, K.S., Mendoza, l., 2003. Immunotherapy for treatment of multicentric cutaneous pythiosis in a dog. J. Am. Vet. Med. Assoc. 223, 215–218. Marin, C., Lopez, N., Perez, C., Mirabal, G., Rolo, M., Urdaneta, J., 1991. Primer reporte de la Granulomatosis Enzootica Bovina en Venezuela. Fonaiap Divulga 37, 15–16. McMullan, W.C., Joyce, J.R., Janselka, D.V., Heitman, J.M., 1977. Amphotericin B for the treatment of localized subcutaneous phycomycosis in the horse. J. Am. Vet. Med. Assoc. 170, 1293– 1298. Mendoza, L., Kaufman, L., Standard, P., 1986. Immunodiffusion test for diagnosis and monitoring pythiosis in horses. J. Clin. Microbiol. 23, 813–816. Mendoza, L., Kaufman, L., Standard, P., 1987. Antigenic relationship between between the animal and human pathogen Pythium insidiosum. J. Clin. Microbiol. 25, 2159–2162. Mendoza, L., Nicholson, V., Precott, J.F, 1992. Immonoblot analysis of the humoral immune response to Pythium insidiosum in horses with pythiosis. J. Clin. Microbiol. 30, 2980–2983. Mendoza, L., Ajello, L., McGinnis, L., 1996. Infections caused by the oomycetous pathogen Pythium insidiosum. J. Mycol. Med. 6, 151–164. Mendoza, L., Kaufman, L., Mandy, W., Glass, R., 1997. Serodiagnosis of human and animal pythiosis using an enzyme-linked immunosorbent assay. Clin. Diagn. Lab. Immunol. 4, 715–718. Mendoza, L., Mandy, W., Glass, B., 2003. An improved Pythium insidiosum-vaccine formulation with enhanced immunotherapeutic properties in horses and dogs with pythiosis. Vaccine 21, 2797–2804. Miller, I.R., 1981. Treatment of equine phycomycosis by immunotherapy and surgery. Aust. Vet. J. 57, 377–382. Miller, R.I., Olcott, B.M., Archer, M., 1985. Cutaneous pythiosis in beef calves. J. Am. Vet. Med. Assoc. 186, 984–986. Reis, J.L., Quiroz de Carvalho, E.C., Nogueira, R.H.G., Lemos, L.S., Mendoza, L., 2003. Disseminated pythiosis in three horses. Vet. Microbiol. 96, 289–295. Santurio, J.M., Monteiro, A.B., Leal, A.T., Kommers, G.D., de Sousa, R.S., Catto, J.B., 1998. Cutaneous pythiosis in calves from the Pantanal region of Brazil. Mycopathologia 141, 123–125. Thianprasit, M., 1990. Human pythiosis. Trop. Dermatol. 4, 1–4. Tabosa, I.M., Riet-Correa, F., Nobre, V.M.T., Azevedo, E.O., Reis Jr., J.L., Medeiros, R.M.T., 2004. Outbreaks of pythiosis in two flocks of sheep in Northeastern Brazil. Vet. Pathol. 41, 412–415. Thiatithanyanont, A., Mendoza, L., Chuansumrit, A., Pracharktam, R., Laothamatas, J., Sathapatayavongs, B., Lolekha, S., Ajello, L., 1998. Use of an immunotherapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum. Clin. Infect. Dis. 27, 1394–1400. Thomas, R.C., Lewis, D.T., 1998. Pythiosis in dogs and cats. Compendium 20, 63–75.
128
R.C. Pe´ rez et al. / Veterinary Microbiology 109 (2005) 121–128
Vanittanakom, N., Supabandhu, J., Khamwan, C., Praparattanapan, J., Thirach, S., Prasertwitayakij, N., Louthrenoo, W., Chiewchanvit, S., Napaporn Tananuvat, N., 2004. Identification of emerging human-pathogenic Pythium insidiosum by serological and molecular assay-based methods. J. Clin. Microbiol. 42, 3970–3974.
Wanachiwanawin, W., Mendoza, L., Visuthisakchai, S., Mutsikapan, P., Sathapatayavongs, B., Chaiprasert, A., Suwanagool, P., Manuskiatti, W., Ruangsetakit, C., Ajello, L., 2004. Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans. Vaccine 22, 3613– 3621.