Identification of major allergens of Malassezia pachydermatis in dogs with atopic dermatitis and Malassezia overgrowth

Abstract We have previously shown that both atopic and normal dogs generate an IgG response to antigens of Malassezia pachydermatis . The aim of this study was to compare IgE responses to separated proteins of M. pachydermatis in 28 atopic dogs with Malassezia dermatitis and 22 clinically normal dogs using Western immunoblotting. Six different detection systems were evaluated in order to assess sensitivity and eliminate nonspecific binding and cross-reactivity. The protocol yielding the best results utilized a monoclonal mouse antidog IgE, an alkaline phosphatase conjugated goat antimouse IgG which had been passed through a canine IgG column 3 times, a chemiluminescent substrate and a digital imaging system. Proteins of 45, 52, 56 and 63 kDa were recognized by more than 50% of the atopic dog sera and thus represented major allergens. Only a minority of normal dogs showed faint IgE binding to these proteins. The results indicate that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease.     

Response to Malassezia pachydermatis by peripheral blood mononuclear cells from clinically normal and atopic dogs

OBJECTIVE: To investigate the potential cell-mediated immune response of atopic dogs to the yeast Malassezia pachydermatis and to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs. ANIMALS: 16 clinically normal dogs, 15 atopic dogs with Malassezia dermatitis, 5 atopic dogs with Malassezia otitis, and 7 atopic control (ie, without Malassezia dermatitis or otitis) dogs. PROCEDURE: A crude extract of M pachydermatis was extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells. RESULTS: Atopic dogs with cytologic evidence of Malassezia dermatitis had an increased lymphocyte blastogenic response to crude M pachydermatis extract, compared with clinically normal dogs and dogs with Malassezia otitis. Atopic control dogs did not differ significantly in their responses from atopic dogs with Malassezia dermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M pachydermatis within any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-mediated and humoral reactivities to M pachydermatis contribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response toward M pachydermatis may assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs.     

Humoral measurement of type-1 hypersensitivity reactions to a commercial Malassezia allergen

Malassezia pachydermatis is considered to be a contributing factor to canine atopic dermatitis (AD). The purpose of this study was to investigate the humoral response to a commercially produced M. pachydermatis extract. Fifteen atopic dogs with Malassezia overgrowth on the skin (MD), 16 atopic dogs without MD, three atopic dogs with overgrowth of Malassezia in the ears only (MO), and 12 normal dogs were intradermally tested with M. pachydermatis extract at 50, 100, 250, 500, 1000, 2000 and 4000 PNU mL(-1). All dogs were evaluated cytologically by cutaneous tape strip and bilateral ear exudate sampling to determine presence of MD or MO. Each had serum evaluated for anti-Malassezia IgE using three Malassezia extracts with an ELISA assay. The irritant threshold concentration at which healthy nonatopic dogs ceased to react was 1000 PNU mL(-1). There was a significant difference in intradermal test reactivity between the atopic groups. At this dilution, 93% (14/15) of the atopic MD group, 31% (5/16) of the atopic group without MD or MO, and 100% (3/3) of the atopic MO only group reacted. There were no significant differences in the serum IgE levels as measured by the Greer ELISA assay, between any groups using any of the three extracts. These results support that Greer's M. pachydermatis extract is useful for intradermal testing of dogs with an allergic phenotype, and that atopics with MD are more likely to have a type-1 Malassezia hypersensitivity than those without. The ELISA assay may require further development in order to be useful for the diagnosis of Malassezia hypersensitivity.     

Serum antibodies to Malassezia yeasts in canine atopic dermatitis

Significant numbers of humans with atopic dermatitis develop Malassezia-specific IgE. Immediate skin-test reactivity to Malassezia has been demonstrated in atopic dogs. The aim of this study was to compare the serum IgG and IgE response to Malassezia in atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis, nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis and healthy dogs. Cytology was used to diagnose clinically significant Malassezia dermatitis and otitis. Contact plate cultures confirmed the validity of this technique. Reproducible enzyme-linked immunosorbent assays for Malassezia-specific IgG and IgE in canine serum were established. Atopic dogs had significantly higher serum IgG and IgE levels than either healthy dogs or nonatopic dogs with clinical evidence of Malassezia dermatitis and/or otitis. There was no significant difference in IgG and IgE levels between atopic dogs with and without clinical evidence of Malassezia dermatitis and/or otitis. The implications of these findings in the pathogenesis and management of canine atopic dermatitis are discussed.     

Evaluation of serum obtained from atopic dogs with dermatitis attributable to Malassezia pachydermatis for passive transfer of immediate hypersensitivity to that organism

OBJECTIVE: To determine the functionality of canine anti-Malassezia IgE via the passive transfer of immediate hypersensitivity localized to the skin (ie, cutaneous anaphylaxis) from atopic dogs with dermatitis attributable to overgrowth of Malassezia pachydermatis (Malassezia dermatitis [MD]) to healthy recipient dogs by use of the Prausnitz-Küstner (P-K) technique. ANIMALS: 7 clinically normal dogs, 32 atopic dogs with MD, serum from 11 atopic dogs with MD, and 3 healthy dogs without prior sensitization to M pachydermatis. PROCEDURE: Serum from atopic dogs with MD was used for P-K tests in 3 clinically normal recipient dogs. Serial dilutions of untreated, heat-inactivated, IgE-absorbed, and bovine serum albumin (BSA)-absorbed (control) aliquots of serum were injected ID in triplicate for dermal sensitization. Twenty-four, 48, and 72 hours later, a crude extract of M pachydermatis was injected ID into the sites used for sensitization injections, and immediate hypersensitivity reactions were graded on a 4-point scale. RESULTS: Untreated serum caused P-K reactivity beginning 24 hours after passive sensitization and persisting through 72 hours (titers, 1:32 to 1:64). Heat inactivation and IgE-absorption of serum eliminated P-K reactivity, whereas treatment of serum with BSA did not. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of P-K test results supports the passive transfer of cutaneous anaphylaxis by anti-Malassezia IgE and indicates it is functional in type-1 hypersensitivity reactions of atopic dogs with MD. Reduction or blockade of anti-Malassezia IgE in atopic dogs with MD may provide better clinical control of the disease.     

Occurrence, distribution and population size of Malassezia pachydermatis on skin and mucosae of atopic dogs

Aim of the present study was to determine the distribution and quantification of Malassezia yeasts on a wide number of cutaneous sites in atopic dogs by means of a semiquantitative swab technique. A possible relationship between the presence of clinical signs and the occurrence and population size of yeasts was attempted. Forty-one privately owned atopic dogs of different age and breed were sampled. Results were expressed as colony forming units per swab. Malassezia colonies obtained from each plate were counted, scored and typed. All dogs yielded Malassezia pachydermatis from at least one skin area. Yeast population mean size by site was 6.98 (S.D.=3.47) as compared to other body areas. The frequence of isolation was higher from interdigital areas (70.7%), ears (63.4%), nail folds (35.7%), mouth (33.3%), groin (30.9%), conjunctiva and axillae (23.8%), perineum and anus (19%), perianal glands (9.5%). Ears, anus, interdigital areas, perianal glands and groin yielded the largest mycotic amount. M. pachydermatis was the sole species of yeast to colonize canine skin in examined animals. No statistical correlation between the presence of cutaneous alterations and Malassezia isolation was detected. Highest scores were not exclusively found on affected areas, but also on lesion-free sites, demonstrating that atopic animals can be heavily colonized also in apparently healthy areas.     

Genotyping of Malassezia pachydermatis isolates from canine healthy skin and atopic dermatitis by internal spacer 1 (IGS1) region analysis

Isolates of Malassezia pachydermatis from healthy dog skin and from dogs with atopic dermatitis were molecularly characterized using internal spacer 1 (IGS1) region analyses, and their phospholipase A2 activity and pH growth profiles were then characterized in vitro. The percentage of isolates from healthy dogs that had the following IGS1 subtypes (isotype, %) were as follows: 1A, 6%; 1B, 27%; 1C, 11%; 2A, 6%; 2B, 6%; 3A, 11%; 3C, 3%; and 3D, 24%. In contrast, 9% of isolates from dogs with atopic dermatitis were isotype IB and 91% were isotype 3D, indicating that isolates of subtype 3D were the most prevalent in dogs with atopic dermatitis. Production of phospholipase A2 was statistically higher in isolates of subtype 3D than in the other subtypes. The subtype 3D isolates showed enhanced growth on alkaline medium compared with non-3D subtype isolates. The main clinical sign of canine Malassezia dermatitis is waxy exudates on the skin, which predispose the patient to development of a yeast overgrowth of the subtype 3D. Increased phospholipase A2 production may be involved in the inflammatory process associated with Malassezia dermatitis.     

Malassezia pachydermatis isolated from normal and diseased external ear canals in dogs: a comparative analysis

To investigate the role of Malassezia pachydermatis as a pathogenic agent in canine otitis, a comparative analysis of isolates from normal and diseased external ear canals in dogs was undertaken. Specimens were collected from the ears of dogs with unilateral or bilateral otitis and from healthy dogs. Mycological analysis was by direct microscopy and fungal culture on Sabouraud's dextrose agar and Dixon's agar. Of the otitis specimens, 63.7% showed typical Malassezia cells on cytological examination. In samples taken from the healthy ears of dogs with unilateral otitis, only 21.43% (P<0.05) showed evidence of Malassezia. M. pachydermatis was identified cytologically and culturally in 57.53% (P<0.05), 14.29% and 30.0% of samples from the ears of dogs with otitis, from the healthy ears of dogs with unilateral otitis and from the ears of healthy dogs with no otitis. In the group with otitis associated with M. pachydermatis, the poodle was the most common breed (39.29%; P<0.05), whereas in the group without otitis, the German Shepherd breed was prominent (although this observation was not statistically significant). In both groups, the majority of dogs with M. pachydermatis were aged between 1 and 3 years (P<0.05). The higher incidence of M. pachydermatis isolated from the ears of dogs with otitis externa suggests a putative pathogenic role of this yeast in this condition.     

Assessment of the ability of Malassezia pachydermatis to stimulate proliferation of canine keratinocytes in vitro

OBJECTIVE: To investigate the direct interaction between canine keratinocytes and live Malassezia pachydermatis and thereby determine the role of these organisms in the pathogenesis of epidermal hyperplasia associated with Malassezia dermatitis in dogs. SAMPLE POPULATION: Primary canine keratinocyte cultures established from skin samples obtained from clinically normal dogs. PROCEDURE: The proliferative response of keratinocytes co-cultured with Malassezia organisms for 1, 2, or 3 days was assessed by use of direct manual counting (to determine the number of keratinocytes in both the monolayer and the medium) and immunohistochemical staining techniques involving antibodies against proliferating cell nuclear antigen (PCNA) and another cellular proliferation marker, Ki-67. The potential cytotoxic effect of Malassezia organisms was investigated by use of an apoptosis detection kit to label keratinocytes co-cultured with M. pachydermatis that underwent apoptosis. RESULTS: No stimulatory effect of Malassezia organisms on canine keratinocyte proliferation was detected via cell counting and immunohistochemical techniques. However, there was a significant increase in dead keratinocytes in the medium with increasing numbers of Malassezia organisms in the co-culture. More apoptotic cells were observed in keratinocyte monolayers co-cultured with high numbers of M. pachydermatis than there were in monolayers cultured without Malassezia organisms, and the number increased after prolonged incubation. CONCLUSIONS AND CLINICAL RELEVANCE: M. pachydermatis did not stimulate canine keratinocyte proliferation in vitro. The results suggested that the epidermal hyperplasia observed in dogs with Malassezia dermatitis is unlikely to be caused by a direct effect of the organism on the keratinocyte cell cycle, but is likely to involve other mechanisms.     

In vitro antifungal susceptibility of Malassezia pachydermatis from dogs with and without skin lesions

Canine Malassezia dermatitis is frequently treated with systemic ketoconazole (KTZ) and itraconazole (ITZ). However, no information is available on the antifungal susceptibility to azoles and allilamine of Malassezia pachydermatis isolates from dogs with or without skin lesions. The present study was designed to evaluate the in vitro antifungal susceptibility of M. pachydermatis strains from dogs with or without skin lesions to KTZ, ITZ, miconazole (MICO), fluconazole (FLZ), posaconazole (POS), voriconazole (VOR) and terbinafine (TER) using the Clinical and Laboratory Standards Institute reference Broth Microdilution Method (CLSI M27-A2). The association between the susceptibility to antifungal compounds and the origin of M. pachydermatis, from skin with or without lesions has been also assessed. A total of 62 M. pachydermatis strains from healthy dogs (i.e., Group A=30) or with skin lesions (i.e., Group B=32) were tested. ITZ, KTZ and POS showed the highest activity against M. pachydermatis strains, whereas MICO TER and FLZ the lowest. A higher number of Malassezia resistant strains were registered among isolates from Group B than those from Group A. This study indicates that M. pachydermatis strains were susceptible to ITZ, KTZ, and POS. However, dogs with lesions may harbour strains with low susceptibility to antifungal agents and displaying cross-resistance phenomena to azole. The antifungal therapy in Malassezia infections requires careful appraisal of choice of drugs especially in cases of unresponsiveness to antifungal treatment or recurrent infections.     

Comparison of two sampling techniques for the detection of Malassezia pachydermatis on the skin of dogs with chronic dermatitis

Adhesive tape strip and dry swab sampling techniques were compared for the detection of Malassezia pachydermatis on the skin of dogs with chronic dermatitis. One hundred and four dogs were sampled by each of the techniques. Two methods, a culture method and a stain method, were used to assess the sampling techniques. By the adhesive tape strip sampling technique, M. pachydermatis was detected on 83 (80%) dogs using the culture method and on 45 (43%) dogs using the stain method. By the dry swab sampling technique, M. pachydermatis was detected on 55 (53%) dogs using the culture method and on 33 (32%) dogs using the stain method. The study showed that the adhesive tape strip sampling technique, using the culture method, detected Malassezia on the skin of significantly more dogs (P<0.001) than the same technique using the stain method and also significantly more than the dry swab sampling technique, using either the culture or stain methods. It was also shown that an adhesive tape sample could be used to transfer cells to a slide for staining and microscopy prior to being used for culturing Malassezia.     

Higher incidence of Malassezia pachydermatis in the eyes of dogs with corneal ulcer than in healthy dogs

Malassezia pachydermatis is usually associated with otitis and dermatitis in dogs but it can also cause diseases in other species, including humans. In a human neonatal intensive care unit, M. pachydermatis was isolated from an infant's ocular discharge. Therefore, the aim of this study was to ascertain the presence of Malassezia spp. and its possible consequences in dogs' eyes. This research included 19 dogs with unilateral or bilateral corneal ulcers and 60 healthy dogs. A total of 158 clinical specimens from both the groups were obtained from the conjunctival sac of each eye by a calibrated platinum loop. The samples were placed on Dixon and blood agar, incubated at 35 degrees C, and examined daily for 15 days. Then, the strains were subcultured on Sabouraud agar. Of 22 clinical specimens collected from the eyes with corneal ulcers, five cultures (23%) were positive for M. pachydermatis. Of 16 samples collected from the contralateral healthy eye, cultures were positive in three samples (19%). Three animals had unilateral corneal ulcer and positive cultures for M. pachydermatis in both the eyes. Two dogs had unilateral corneal ulcer and positive cultures for M. pachydermatis in the same eye. However, from the 120 samples of 60 healthy dogs, only four clinical specimens (3%) had positive cultures for M. pachydermatis. The findings of M. pachydermatis, in a considerable percentage of clinical specimens from dogs with corneal ulcer, suggest its possible role at least as an aggravating factor in the pathophysiology of this disease.     

Frequency, body distribution, and population size of Malassezia species in healthy dogs and in dogs with localized cutaneous lesions.

Malassezia species are commensal organisms of human and animal skin that occasionally act as opportunistic pathogens. The lipid-dependent species are associated with human skin disorders, whereas the non-lipid-dependent species (Malassezia pachydermatis) is considered as an opportunistic secondary pathogen affecting the canine skin surface and ear canal. This study evaluated the relationship between Malassezia yeasts, their population size, and the occurrence of skin lesions from healthy and skin-diseased dogs. The efficiency of cytological examination and fungal culture for Malassezia detection was also evaluated. From March 2002 to July 2003, 33 healthy dogs and 54 dogs with pruritic localized skin diseases were examined; skin swabs (1218) were collected from 7 anatomical sites for culture and cytological examination. Malassezia prevalence according to anatomical site and the agreement between cytological results and fungal cultures were statistically analyzed. Differences in mean colony forming unit counts between positive healthy and diseased dogs were evaluated using the Bonferroni test for post hoc pair-wise comparisons. In healthy dogs, Malassezia yeasts were most frequently isolated in the perianal and perioral areas. The frequency of isolation and population size of Malassezia species were higher in dogs with localized dermatitis, especially in affected areas, indicating a role for Malassezia in the occurrence of skin lesions. Malassezia pachydermatis was the species most commonly cultured from the skin and external ear canal of healthy and diseased dogs; isolation of lipid-dependent yeasts from healthy dogs was less frequent. Using fungal culture as the gold standard, cytological examination showed good relative specificity (95%) but very low relative sensitivity (30%).     

Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa

The bacterial and fungal flora of the external ear canal of dogs with otitis externa and of healthy dogs were studied. The most frequently isolated microorganism from otitic ears was Staphylococcus intermedius (58.8%), followed by Malassezia pachydermatis (30.9%), Streptococcus canis (29.9%), Proteus spp. (14.4%) and Escherichia coli (10.3%). A statistical analysis of our results showed that the prevalence of these microorganisms is significant in dogs with otitis externa. Furthermore, the antimicrobial susceptibility patterns of isolated strains were determined. Majority of all bacterial isolates were most susceptible to gentamicin. Malassezia pachydermatis, the most prevalent yeast in this study, showed an excellent level of susceptibility to all antifungal agents tested.     

Comparative analysis of the frequency, distribution and population sizes of yeasts associated with canine seborrheic dermatitis and healthy skin

The purpose of this study was to investigate the diversity of yeast associated with the degree of canine seborrheic dermatitis (SD) by anatomical sites. Fifty-seven samples were divided as 17 healthy skin, 20 with primary seborrheic dermatitis (PSD), and 20 with secondary seborrheic dermatitis (SSD). Yeast isolation and characterization were carried out based on microscopical features and biochemical properties. DNA analysis at the internal transcribed spacer I of 26S rDNA region was utilized for species confirmation. Four species of yeast consisting Malassezia pachydermatis, Malassezia furfur, Candida parapsilosis and Candida tropicalis recovered from examined dogs. M. pachydermatis and C. parapsilosis were isolated from all dogs, but C. tropicalis and M. furfur were recovered from 3 healthy dogs and one diseased dog, respectively. The number of M. pachydermatis and C. parapsilosis in diseased dogs was higher than that of healthy specimens (P<0.01). High frequency and population size of C. parapsilosis were closely associated to PSD, while those of M. pachydermatis were associated with both PSD and SSD (P<0.01). C. parapsilosis were predominant at the perianal area. This study demonstrated the co-colonization of M. pachydermatis and C. parapsilosis in large amounts and frequency associated with stage of disease and anatomical site.     

Mycologic disorders of the skin

Cutaneous tissue can become infected when fungal organisms contaminate or colonize the epidermal surface or hair follicles. The skin can be a portal of entry for fungal infection when the epithelial barrier is breached or it can be a site for disseminated, systemic fungal disease. The two most common cutaneous fungal infections in small animals are dermatophytosis and Malassezia dermatitis. Dermatophytosis is a superficial cutaneous infection with one or more of the fungal species in the keratinophilic genera Microsporum, Trichophyton, or Epidermophyton. Malassezia pachydermatis is a nonlipid dependent fungal species that is a normal commensal inhabitant of the skin and external ear canal in dogs and cats. Malassezia pachydermatis is the most common cause of Malassezia dermatitis. The diagnosis and treatment of these cutaneous fungal infections will be discussed.     

Frequency of yeasts and dermatophytes from healthy and diseased dogs.

The aim of this study was to investigate the presence of dermatophytes and yeasts in healthy and diseased dogs. A total of 633 samples were collected from 26 healthy animals (104 samples), 131 with dermatitis (343 samples), 74 with otitis (148 samples), and 19 with ocular diseases (38 samples). Cultures from healthy animals were positive for Malassezia pachydermatis in 13.5% (7/52) of samples from skin, 42.3% (11/26) from ear, and 3.8% (1/26) from eye. Fungal growth was observed in 20.4% (70/343) samples from animals with dermatitis. Microsporum canis was the most isolated fungus (n = 39), followed by M. pachydermatis (n = 30) and Malassezia sp. (n = 3). Of the 148 samples from dogs with otitis, 90 (60.8%) were positive for M. pachydermatis, and of the clinical specimens from the conjunctiva of animals with ophthalmic disease, 2.6% (1/38) presented positive cultures for M. pachydermatis. Only 14.3% (2/14) of the positive cultures for M. pachydermatis and 40.9% (9/22) of those for M. canis were positive in the direct exam. Direct exams were positive in 84.3% (70/83) of the culture positive samples from affected ears of dogs with otitis. Malassezia pachydermatis may act as an aggravating factor in the occurrence of cutaneous diseases, or the isolation of M. canis may be associated with the onset of dermatophytosis. Fungal culture, rather than microscopic examination, should be used as the definitive diagnostic test for dermatomycoses and otitis.     

Chromosome-sized DNA of Malassezia pachydermatis by pulsed-field gel electrophoresis.

The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes by using pulsed-field gel electrophoresis and their molecular sizes were calculated as 820, 1,100, 1,400, 1,470, 1,660 and 1,820 Kb, respectively. Comparison of electrophoretic patterns suggested that the chromosomes of M. pachydermatis were homozygous.     

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.     

Colonisation status of Malassezia pachydermatis on the hair and in the hair follicle of healthy beagle dogs

Hair and hair follicle carriage of Malassezia pachydermatis was studied in 12 healthy beagle dogs. The yeast was isolated from hair clipped from the lip region at 13 sites in nine dogs but was less frequently recovered from the interdigital spaces on the forefeet and from two sites on the trunk. Population sizes at the lip were significantly greater (P < 0.01) than those at other sites. Skin biopsy specimens were obtained from the same sites and epidermal and follicular tissues dissected following immersion in 1 M CaBr(2). Epidermal carriage of M. pachydermatis was identified in nine biopsy specimens taken from five dogs. Hair follicle carriage was identified in five skin specimens (four foot, one lip) from three dogs. This study indicates that M. pachydermatis is readily recovered from the distal hair in healthy dogs and that hair follicle carriage is infrequent or that populations are low at that site.