Chlorella Infection in a Sheep in Mexico and ... - Springer Link

Mycopathologia (2010) 169:461–466 DOI 10.1007/s11046-010-9287-4

Chlorella Infection in a Sheep in Mexico and Minireview of Published Reports from Humans and Domestic Animals R. Ramı´rez-Romero • L. E. Rodrı´guez-Tovar A. M. Neva´rez-Garza • A. Lo´pez

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Received: 27 October 2009 / Accepted: 5 February 2010 / Published online: 18 February 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Algal infections are rare in humans and domestic animals. Prototheca spp. and Chlorella spp. are among the most commonly reported. Herein, we present a brief review on Chlorella spp. infections and related pathologies and discuss this information including a natural case in a sheep in Mexico with a disseminated form of the disease. Keywords Sheep

Chlorella Disseminated infection

Introduction Protothecosis and chlorellosis are rare pseudofungal diseases in humans and animals caused by the opportunistic algae of the Chlorellaceae family, Prototheca spp. (achlorophyllous mutant) and Chlorella spp. (chlorophyll-containing green alga), respectively [1, 2]. Prototheca spp. is more frequently R. Ramı´rez-Romero L. E. Rodrı´guez-Tovar (&) A. M. Neva´rez-Garza Cuerpo Acade´mico de Patobiologıá, Facultad de Medicina Veterinaria y Zootecnia, UANL, Ave. Francisco Villa, s/n, Col. Ex Hacienda El Canada´, Gral. Escobedo, NL 66050, Mexico e-mail: [email protected] A. Lo´pez Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada

reported than Chlorella spp. both in humans and domestic animals [3, 4]. Chlorella spp. has been referred as causing infection in one sole case in humans [5]. Except for sheep and cattle, in which chlorellosis has been recognized more frequently, reports in animals are scarce [4]. Herein, we present a brief review on Chlorella spp. causing infections and other related pathologies in humans and animals, including the description of a new case in a sheep in Mexico. In humans, protothecosis is classified into three main clinical forms: localized cutaneous, articular (olecranon bursitis is the most common), and disseminated infection involving many internal organs [1, 2]. The latter has been clearly associated with an altered immune response [1, 3, 6, 7]. In veterinary medicine, protothecosis is most frequently associated to bovine mastitis [3, 8], but it is also described as localized or disseminated infections in goats (cutaneous) [9], dogs (cutaneous, enteric, ocular, and disseminated), and cats (cutaneous) [10, 11]. Chlorella spp. cause localized lesions such as those described in a woman with a contaminated surgical wound [5]. However, in animals, some cases progress into a disseminated systemic infection [4]. Chlorellosis has been documented in sheep [12–15], cattle [16, 17], dromedary [18], beaver [19], gazelle [20], and most recently, in a dog [4]. This information is resumed in Table 1. The life cycles of Chlorella spp. and Prototheca spp. are similar and involve asexual internal septation

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Mycopathologia (2010) 169:461–466

Table 1 Reported cases of green algae infections in humans and animals with the correspondent pathogenesis Cases

Infections

Pathogenesis

Human

Localized cutaneous

Surgical wound contaminated in a river

[5]

Sheep

Localized lymph nodes or disseminated systemic

Probably enteric infection due to drinking stagnant water, sewage water and/or grazing pastures irrigated with nontreated sewage water

[11–14]

Cattle

Localized lymph nodes

Probably enteric infection and posterior location on lymph nodes. Most cases recognized at inspection

[15, 16]

Gazelle

Localized cutaneous

Local contamination probably

[19]

Dromedary

Enteric

Enteric infection due to drinking stagnant water and/or grazing pastures irrigated with non-treated sewage water

[17]

Beaver

Localized cutaneous

Subcutaneous contamination probably

[18]

Dog

Disseminated systemic

Enteric infection and subsequent systemic dissemination

[4]

by cytoplasmic cleavage of the parent cell. The process is called endosporulation and the parent cell sporangium. Each sporangium cell contains 2 to 20 sporangiospores approximately. Once cell maturation has been completed, the thick wall of the sporangium ruptures releasing sporangiospores into the environment to complete the life cycle [1, 2]. Algae are ubiquitous in nature and infections are considered opportunistic [1–3]. In the case of Chlorella spp., infections are favored by contamination of wounds in humans [5] and animals [20]. Other infections are thought to happen after dissemination from gastrointestinal tract in immune compromised animals [4]. In sheep, infections are disseminated and occurred via digestive tract when animals are grazing lands irrigated with contaminated sewage water from canals [13–15]. Table 1 shows a list of cases reported with the probable pathogenesis. Prototheca spp. and Chlorella spp. induce commonly a strong granulomatous reaction and both organisms are morphologically similar in paraffinembedded sections stained with H&E. Typically, these algae have oval to round shape, vary in size from 8 to 30 lm in diameter and have a PAS-positive thick capsule. One important microscopic difference is the presence of abundant PAS-positive starch granules in the cytoplasm of Chlorella spp. Another remarkable feature that can be detected by gross observation is that in chlorellosis the affected tissues have a distinctive green discoloration [1, 2, 4, 5, 11, 16, 17, 20–24] that remains obvious even after formalin fixation, a feature not seen in protothecosis [15]. However, the differential diagnosis is ultimately

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Reference

confirmed by transmission electron microscopy where chloroplasts and starch bodies are present in Chlorella spp. but absent in Prototheca spp. [2, 4, 13, 16, 20, 21]. Table 2 resumes these characteristics.

Case Report Formalin-fixed tissues from a dead ewe were submitted to the Veterinary Diagnostic Laboratory of the Universidad Autońoma de Nuevo Leoń for histopathological examination. These tissues were from a 48-month-old ewe with accompanying medical history of progressive emaciation. At the final stage, the owner had also noticed anxiety and discomfort characterized by a persistent looking to their flanks and kicking on the ground. Occasionally, the ewe fell down over her belly shaking her head vigorously. Following death, the farmer opened the body cavities and noticed froth in bronchi, mottled appearance of the liver and enlargement of visceral lymph nodes, some of which had ‘‘abscess-like lesions.’’ This ewe belonged to a 200 heads flock in Piedras Negras, Coahuila, Mexico, about 0.75 miles south from the border city of Eagle Pass, Texas, USA. According to the owner, eight other animals ranging in age from young to adults had died or were killed in extremis because of progressive emaciation during September and October, 2008. Some animals (about 20) had diarrhea and had been unsuccessfully treated with antibiotics (Oxytetracycline, 2 ml/10 kg; EngemycinÒ Intervet Me´xico, S.A. de CV.) and vitamins (ADE, 2 ml; Vigantol ADE Fuerte, Bayer de Me´xico,


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Table 2 Diagnostic differences between Prototheca spp. and Chlorella spp. Characteristics

Prototheca spp.

Chlorella spp.

Gross pathology, color of the lesions

Multinodular granulomatous reaction colorless or non-specific yellowish discoloration [1, 2, 9–11]

Multinodular granulomatous reaction and characteristic greenish and bright (emerald-like) discoloration in fresh tissues [1, 2, 4, 5, 11, 16, 17]. Greenish color remains even after formalin fixation [reference 10 and Fig. 1]

Histopathology, H&E stain

Similar findings and considerations for Prototheca spp. and Chlorella spp. Multinodular granulomatous or pyogranulomatous chronic lesion with intralesional algal organisms. Macrophages and giant cells contain most of the algal cells intracytoplasmic. These algal cells are large single or binucleated cells (sporangium) as well as septated cells (endosporulation), 8–15 lm in diameter. In addition, similar or larger morula-like cells, 10–30 lm diameter, containing 4, 8 or more spores within (sporangiospores), may seen admixed. In sites with a strong inflammatory response and necrosis is common to see empty shell-like and refractile structures [1, 2, 4, 5, 9, 11–24]. Occasionally, inflammatory response is minimal [1, 2, 19]

Histopathology, Similar findings and considerations for Prototheca spp. and Chlorella spp. In comparison with Coccidioides other differentials immitis and Rhinosporidium seeberi fungi, algal sporangia are smaller and contain fewer spores. Prototheca spp. and Chlorella spp. also differ from Blastomyces dermatitidis and Cryptococcus neoformans by the size, internal structure and lack of budding [1, 4, 17, 23, 24] Histochemistry, Prototheca spp. has a cell wall that reacts positive [4, Chlorella spp. has cell wall positive as well as distinctive PAS and Grocott 11, 21]. Intracellular components are poorly and strong positive intracytoplasmic granules that stains stained. sometimes occlude completely the cell. Empty shells appear thicker and also strong positive [1, 2, 4, 5, 11– 13, 15–17, 19–24] TEM peculiarities

Chloroplasts and starch bodies absent [1, 2, 21]

S.A. de CV.). Interestingly, the farmer reported that his entire flock had been drinking and grazing in a new improved area irrigated with non-treated sewage water. Gross examination of the fixed tissues revealed that the mesenteric lymph nodes were enlarged and showed coalescing areas of caseous necrosis on cut surface. The liver showed a zonal pattern and had focal to coalescing foci of pale discoloration. The fixed lung appeared mottled and firm. A striking finding observed while trimming the samples was a conspicuous green discoloration of all fixed specimens (Fig. 1). Microscopically, the liver had focal aggregates of large macrophages admixed with few lymphocytes and neutrophils. Most macrophages contained yeastlike structures (Fig. 2) and the whole lesion was often surrounded by variable amounts of fibrous connective tissue. The intracellular organisms were round to oval, 8–14 lm in diameter and had a thick refractile cell wall that was more evident when the cysts were empty. Occasional binucleation was observed. Sporulated organisms were larger contained 2–8 spores, ranging from 2 to 6 lm in diameter (Fig. 2). Microscopic lesions in the lymph nodes were similar, but

Presence of chloroplast structures such as grana (stacks of membranes) and associated thylakoid membranes as well as starch bodies embedded [1, 2, 4, 13, 18, 20, 21]

Fig. 1 Formalin-fixed tissues: A lung, B lymph node, and C liver. Note green discoloration in all tissues. Bar = 2.5 cm

necrosis was more extensive with abundant empty and refractile shells. In the lung, the lesions and the organisms were less numerous but still easy to find. The organisms had a strongly PAS-positive cell wall and abundant positive dense granules in the cytoplasm. The cell walls and granules also stained strongly positive with the Grocott’s stain (Fig. 3). Ultrastructural identification of the alga was done by transmission electron microscopy. The cell wall appeared as a thick electron-lucent structure, but the

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Fig. 2 Liver. Granuloma formed by large macrophages containing organisms in different stages of development. Sporangium (arrows) and endospores (arrowheads). Fibrous connective tissue admixed with cellular infiltrates, mostly macrophages and few lymphocytes. H&E stain. Bar = 20 lm Fig. 4 Liver. Single algal organism showing a thick cell wall (CW), large chloroplasts with thylakoid-stacks (grana) (arrows) of different size, and electron-dense (osmiophilic) starch granules (S). There are also multiple lipid droplets (arrowheads). TEM; uranyl acetate and lead citrate. Bar = 2 lm

Fig. 3 Lung. Large number of organisms showing dense positive granules in the cytoplasm. Grocott stain. Bar = 20 lm

most streaking feature was the presence of small and large chloroplasts with thylakoid-stacks (grana) and electron-dense (osmiophilic) starch granules (Fig. 4). Chloroplasts and starch granules were larger in the sporulated cells. Other ultrastructural findings include translucent cytoplasmic lipid droplets and organisms with 2 or rarely 3 nuclei.

Discussion Diagnosis of chlorellosis is largely based on morphological identification of the organisms by special stains and electron microscopy. In this report, the

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green color of the lesions, the presence of intralesional algae in granulomas, the abundant PAS-positive granules in the cytoplasm, and the characteristic chloroplasts and starch granules demonstrated by TEM supported the diagnosis of ovine chlorellosis. These criteria have been considered fundamental to establish Chlorella spp. diagnosis [4, 20, 21]. To our knowledge, this is the first documented case in Mexico. It is worth to mention that chlorellosis in animals might be caused by different algae of the family Chlorellaceae [13, 20]; however, Cordy’s first report of green algal infection in a sheep [12] influenced the terminology. Chlorellosis is a rare disease most frequently reported in sheep [7–10]. In cattle, the infection was referred as incidental findings of affected lymph nodes at slaughterhouse inspection [16, 17]. There are still some unanswered questions regarding the natural transmission of Chlorella spp. in domestic animals, but ingestion of stagnant water contaminated with sewage or pastures irrigated with raw sewage is considered the most common route of infection in sheep [8–10]. Oral route is also the most likely route of infection in cattle [16, 17], as well as a dromedary [18] and a dog [4].


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Table 3 Other pathologies related with green algae in humans and the correspondent pathogenesis Related pathologies

Pathogenesis

Reference

Interstitial nephritis

Hypersensitivity reaction due to consumption of supplement

[30]

Cutaneous erythema

Accumulation of chlorophyll-related compounds and subsequent photosensitivity

[31]

Asthma (occupational) Allergy

Hypersensitivity to Chlorella’s powder IgE-related hypersensitivity

[32] [33]

Chlorella spp. has been referred as an opportunistic organism that often disseminates from the gastrointestinal tract to other organs in immunosuppressed animals [4, 12, 18]. Nonetheless, we have no arguments to suppose an altered immune response in this ewe. Interestingly, progressive emaciation and diarrhea were obvious in some ewes in the flock, and these clinical signs were reported previously in sheep with disseminated infections [13, 15]. The algae in this ewe were observed in liver, lymph nodes and lung, and most likely would be present in other tissues that were not submitted. On this basis, we consider the case herein reported as disseminated chlorellosis. Chlorella spp. has been proposed as a rich-one nutritional supplement in ruminants [25]. Nutritional benefits have also been demonstrated employing Chlorella spp. as carrier of organic-linked minerals in pigs and poultry [26]. In humans, Chlorella spp. has been anciently utilized as food source and more recently as nutritional supplement. Chlorella spp. supplements are claimed as exceptional nutriments with additional benefits including, improvement of the immune response, promoter of epithelial healing in small intestine, antioxidant action, and even antitumoral effects [27–29]. Experiments in mice showed benefits in animals with experimental induced inflammation as well as protective effects against cancerigenous drugs [29]. Nonetheless, there are several reports in humans that require judging the potential of Chlorella spp. as a risk of undesirable side effects [30–33]. Table 3 summarizes these conditions. Acknowledgment PIFI, SESIC-SEP.

This work was supported by funds of

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