Common variable immune deficiency in a Pomeranian with Pneumocystis carinii pneumonia.

A Pomeranian dog, 1 year- and 8 month-old neutered female, was presented with persistent respiratory distress and recurrent generalized demodicosis. Physical examination revealed cyanosis, rough respiratory sounds, multifocal alopecia and dermal erosions on the dorsal side of the forelimbs, perineal area and skin around the eyes. A severe diffuse interstitial lung pattern was observed on thoracic radiographs. The blood examination revealed neutrophilia and hypoglobulinemia. Serum immunoglobulin concentrations of IgG and IgA were low. Histopathological examination revealed severe diffuse interstitial pneumonia with Pneumocystis carinii infection. Severe lymphoid depletion was observed in the spleen and other organs with lymphoid follicles consisted mainly of CD3-positive T cells and few cells of B-cell lineage. B-cell hypoplasia with subsequent antibody deficiency was suspected.

Species of the genus Pneumocystis exists as fungal pathogens that cause opportunistic infection and subsequent pneumonia in various species including human [1, 4, 10, 12, 13, 15, 16, 21]. Several reports described naturally infected dogs, some of which showed concurrent immunodeficiency [2, 8, 11, 17, 19]. Here, we report a clinical and pathological description of a Pomeranian with pneumocystis pneumonia, demodicosis and underlying immunodeficiency. Sequence analysis of mitochondrial large-subunit RNA and subsequent phylogenetic tree analysis was also performed. This is the first report of Pneumocystis pneumonia in Pomeranians, and the autopsy findings let us to a new insight into the pathogenesis of Pneumocystis pneumonia in dogs.

A 20-month-old, neutered, female Pomeranian dog was referred to the Veterinary Medical Center of the University of Tokyo for respiratory distress and coughing, which failed to respond to treatment with antimicrobials for 1 month. The dog also had a history of recurrent generalized demodicosis since her birth. Owing to the severe respiratory signs, her appetite and activity had decreased to 10–20% of her usual healthy condition.

Physical examination revealed severe respiratory distress with panting and cyanotic mucous membranes (arterial oxygen saturation was approximately 70% when breathing room air). The patient’s body temperature (38.5°C) and heart rate (120 beats per min) were within the respective reference ranges. The dorsal side of both forelimbs and the perineal area were alopecic, and the skin around the eyes had also developed alopecia and erosions.

Thoracic radiographs showed a severe diffuse interstitial lung pattern with a mild peripheral alveolar lung pattern in focal areas. The main laboratory findings included hypoproteinemia (4.4 g/dl) due to hypoglobulinemia (1.4 g/dl), leucocytosis (31,700/µl) with neutrophilia (24,400/µl) and monocytosis (5,700/µl) and an increased C-reactive protein concentration (9.0 mg/dl; reference range [RR], <1 mg/dl). The absolute number of lymphocytes was within the normal range (1,270/µl). No infectious agents were noted during examination of the skin. Serum immunoglobulin concentrations of IgG and IgM determined by the immunodiffusion method were 3.2 mg/ml (RR, 8.0–16.0 mg/ml) and 1 mg/ml (RR, 0.5–2.0 mg/ml), respectively; the IgA concentration determined with an enzyme-linked immunosorbent assay (ELISA) was 1.1 mg/ml (RR, 1.33–3.14 mg/ml). Infectious pneumonia with an underlying immunodeficiency disorder was suspected, and the dog was hospitalized to provide intensive treatment, including oxygen supplementation and antibiotics (Imipenem/Cilastatin (TIENAM; Merck & Co., Inc., Whitehouse Station, N.J., U.S.A.) 10 mg/kg three times a day and sulfadiazine/trimethoprim (Tribrissen Injection, Kyoritsu, Tokyo, Japan) 25 mg/kg once a day). The patient showed no improvement in respiratory state and died on the third day after being admitted to the hospital.

At necropsy, the lungs were consolidated and weighed 96 g (patient’s body weight, 2.4 kg) with diffuse congestion in all lobes. The superficial lymph nodes (superficial cervical, axillary and inguinal lymph nodes) were atrophic and difficult to detect. Histopathological examination revealed interstitial pneumonia characterized by moderate infiltration of lymphocytes, diffuse mild fibrosis and congestion in the alveolar wall. Foamy macrophages and small yeast-like organisms filled the alveolar and bronchial spaces. The organisms were positive for periodic acid-Schiff (PAS) and Grocott’s methanamine silver (GMS) stains (Fig. 1A–1C). Demodex mites were identified in the hair follicles and were accompanied by a mild infiltration of lymphocytes. The femoral bone marrow was hyperplastic in myeloid and erythroid lineage. Lymphoid cells were euplastic. In the spleen, lymphoid follicle-like structures were observed, but mature germinal centers were absent (Fig. 2A).

Fig. 1.

Hematoxylin and eosin (A), periodic-acid Schiff (B), Grocott (C), anti-CD3 (D), anti-CD20 (E) and anti-Iba-1 (F) immunostaining of the lung. Bar=100 mm. On Hematoxylin and eosin staining, foamy macrophages and small yeast-like organisms filled alveoli and bronchial space with interstitial infiltration of lymphocytes and mild fibrosis. The organisms were positive for periodic-acid Schiff (B) and Grocott (C) stains. Almost all of the infiltrated lymphocytes were positive for anti-CD3 immunostaining (D), and CD20 positive cell was hardly detected (E). Iba-1 positive macrophages were abundant (F).

Fig. 2.

Hematoxylin and eosin and Immunohistochemical staining of the spleen. Bar=500 mm (A), Bar=100 mm (B–H). Clusters of lymphocytes, or follicular-like structure, were seen, but no mature germinal center was observed (A (lower magnification) and B (higher magnification)). CD3- positive lymphocytes were abundant (C), but the number of B-cell lineage (CD20 (D), PAX5 (E), IgG (F) and IgM (G) –positive cells) was markedly decreased. Anti-Iba-1 (H) immunostaining showed lack of dendritic cells that should compose germinal center, suggesting that follicular structure did not form in the spleen.

Immunohistochemistry was done by polymer enhancing system (Envision, Dako, Tokyo, Japan). The details of antibodies are summarized in Table 1. DNA was extracted from a paraffin-embedded block of lung tissue using the commercially available TaKaRa DEXPAT™ Easy kit (Takara Bio Inc., Tokyo, Japan), and PCR was conducted with primers targeting the mitochondrial large-subunit ribosomal RNA (mtLSU rRNA) genes of Pneumocystis carinii (pAZ102-H and pAZ102-E) with GeneAmp® PCR System 2700 (Applied Biosystems, Singapore). An amplicon of approximately 350 bp in size was confirmed with agarose gel electrophoresis. Direct sequence analysis of the PCR product was performed with a DNA sequencing kit (BigDye® Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems, Foster City, CA, U.S.A.), sequenced by a sequencer (3,130 × l Genetic Analyzer, Applied Biosystems, Hitachi, Japan) and analyzed in Neighbor Joining method using MEGA 6.0 [18].

Table 1.

Primary antibodies used in the present study

Antibody against	Host	Dilution	Antigen retrieval	Source
CD20	rabbit	1: 400	no	Thermo Scientific, CA, U.S.A.
CD3	rabbit	1: 100	autoclave	Dako, Tokyo, Japan
IgG	goat	1: 1,000	autoclave	Bethyl Laboratories, TX, U.S.A.
PAX5	rabbit	ready to use	autoclave	Dako, Tokyo, Japan
Iba-1	rabbit	1: 250	autoclave	Wako, Osaka, Japan

Immunohistochemistry for CD20, IgG and PAX5 [22] revealed a markedly decreased number of B lymphocytes and plasma cells in the spleen, lungs, intestine and skin, whereas CD3-positive lymphocytes were abundant in these tissues (Figs. 1D, 1E, 2C, 2D and 2F–2H). In the bone marrow, both B and T cells were not abundant, but were present in a distribution similar to normal dogs. Anti-ionized calcium binding adaptor molecule 1 (anti-Iba-1) immunostaining showed severe infiltration with macrophages in the lungs (Fig. 1F) and a lack of dendritic cells, which should normally compose the germinal centers (Fig. 2F).

The phylogenetic analysis inferred from the mtLSU rRNA sequence comparison demonstrated that P. carinii from dog was different from all previously published P. carinii sequences (Fig. 3) and this phylogenetic tree also indicating that dog-derived P. carinii is most closely related to P. carinii from ferret (Mustela putorius furo). The obtained 329 base pairs of nucleotide sequences were deposited in DNA Data Bank of Japan (DDBJ) under accession number LC009003.

Fig. 3.

Phylogenic tree constructed with mtLSU rRNA sequences of P. carinii from dog and 26 representative human and animal species with DDBJ accession numbers. The number of the scale bar shows the percentage occurrence in 1,000 bootstrap replicates. The isolate reported in this study is indicated in bold. P. c.=P. carinii

This case report describes a Pomeranian with primary immunodeficiency and P. carinii pneumonia, which has been reported in miniature dachshunds [9, 11], Cavalier King Charles Spaniels [8, 19] and several other breeds [2, 6]. We describe the clinical, histological and immunohistochemical aspects of the case with the confirmation of P. carinii by molecular biological analysis. The prominent findings included the marked depletion of B cells in the organs examined and hypoglobulinemia, especially the low concentration of IgG in the serum despite the severe infection.

P. carinii infection, low serum concentrations of IgG and IgA and a decreased number of B cells in the lymphoid tissues of the present case are similar to one report in a miniature dachshund [11]. One difference observed in our case is the later onset of the disease compared to the miniature dachshunds, all of which were under 1 year old when diagnosed. The present case also resembles the case series reported in Cavalier King Charles Spaniels [19], which documented low IgG concentrations and P. carinii infection with demodicosis in several cases. Similar to the present case, most of these dogs were adults.

We were not able to diagnose P. carinii infection prior to the patient’s death, although it was suspected and treatment was initiated. Although tracheal or bronchial fluids should be collected to facilitate the diagnosis [17], it was impossible to perform the recommended diagnostic procedures in our case due to the patient’s severe respiratory distress. The dog responded poorly to treatment with sulfadiazine/trimethoprim, an antibiotic which is reportedly effective against P. carinii infection, possibly due to the severity of the pulmonary lesions [19].

Common variable immunodeficiency (CVID), which is characterized by recurrent infection due to a deficiency in antibody production, is the most common primary immunodeficiency in humans [14, 20]. A variety of molecular mechanisms have been identified in humans, indicating that CVID is a group of heterogeneous diseases and is diagnosed by excluding other identifiable primary immunodeficiencies. Some features of the present case resembled human CVID, such as the adult onset, the immunoglobulin deficiency and the opportunistic infection; however, because the serum IgM concentration of this patient was within the reference range and we did not perform a detailed functional assay, whether this case is truly comparable to human CVID is unknown.

On the basis of the immunohistochemical findings, deficient antibody production was attributed to B-cell hypoplasia or depletion of peripheral immune system. Because lymphocytes identified as B cells were observed in the bone marrow, this dog was capable of producing B cells, but it is postulated that peripheral B-cell maturation or expansion was disturbed by an unknown genetic cause in the present case. The finding of B cell absence in the peripheral immune tissue is similar to the pathological finding in horses with CVID, although B cell was not detected in the bone marrow in horses [5]. This difference can be attributed to the wide spectrum of CVID.

The two pathogens identified in this dog, namely P. carinii and Demodex species, are commonly found in dogs with an underlying immunodeficiency. In dogs, P. carinii infection is usually associated with antibody deficiencies [8, 11, 19], although cellular immunodeficiency has also been reported in a dog with P. carinii infection [6]. Based on these previous descriptions in dogs, the findings in our case and the reports from other species [16], P. carinii infection can be seen in dogs with either a cellular or humoral immune deficiency. B cells and plasma cells were rarely seen surrounding the Demodex-infected hair follicles in this patient. However, these cells were reported to be abundant in the lesions associated with demodicosis in dogs [3, 7], and the absence of antibody-producing cells seemed responsible for the recurring demodicosis in the present case.