Selective IgA deficiency and presumptive polyclonal spike in the beta fraction in a dog with leishmaniosis

A 1-year-old, entire, crossbred, female dog examined for apathy, lethargy, unilateral nosebleed of 2-month duration and the presence of symmetrical ulcerative dermatitis (small ulcers covered by a haemorrhagic crust and surrounded by alopecia on the tip of the ear pinna). Laboratory alterations included anaemia and hyperglobulinemia with a presumptive polyclonal spike in the beta fraction. High antibody levels to Leishmania infantum were detected by enzyme-linked immunosorbent assay. An anti-Leishmania therapeutic protocol was established and a reduction of the anti-Leishmania antibodies was detected by an end point sera dilution enzyme-linked immunosorbent assay during the first weeks of therapeutic protocol, while C-reactive protein concentration was reduced during the period of time when meglumine antimoniate was administered. A good clinical response to the treatment was detected after initiating the anti-Leishmania treatment. Quantitative serology is useful in the short-term, because using a two-fold serial dilution technique correlates with the clinical response.


BACKGROUND
Canine leishmaniosis is a zoonotic disease caused by Leishmania infantum.The most typical clinical signs associated to the disease include lymphadenomegaly, cutaneous lesions, anorexia and ocular lesions. 1 The most typical laboratory alterations detected are an important elevation of total protein and total globulins with serum-protein electrophoresis alteration.Polyclonal gammopathy was the most common electrophoretic pattern observed. 2cute phase proteins (APP) are proteins that change their concentration in serum in response to inflammatory stimuli.These proteins could be divided into positive APP (C-reactive protein, serum amyloid A, haptoglobulin, ceruloplasmin, α2-macrogloublin, α1-acid glycoprotein, fibrinogen and complement) based on an elevation serum concentration or by contrast, negative APP (albumin, transferrin, transthyretin, retinol-binding protein and adiponectin). 3ome of these APPs are considered as biomarkers for monitoring the response to the treatment in different diseases, including canine leishmaniosis.Among APPs, C-reactive protein is the protein with a major increase in serum concentration when signs of inflammation are present in the dog. 4 In canine leishmaniosis, an elevation in C-reactive protein and haptoglobulin has been described in animals with an active infection.
On the other hand, after an anti-Leishmania treatment is administered, a decrease of C-reactive protein can be detected. 5o different therapeutic protocols have been described to treat dog with clinical leishmaniosis, including meglumine antimoniate plus allopurinol or miltefosine plus allopurinol. 6n both protocols, monitoring is recommended during and after anti-Leishmania treatment considering different laboratory parameters such as complete blood count, biochemical profile, serum protein electrophoresis and urinalysis after the first month of treatment and then every 3-4 months.Traditionally, serology is not recommended after 6 months of initial anti-Leishmania treatment, 6 due to the lack of correlation between antibody levels and clinical status improvement. 7his report describes a case of selective IgA deficiency and presumptive polyclonal spike in the beta fraction detected by serum protein electrophoresis associated with L. infantum infection with a good response to treatment with meglumine antimoniate and allopurinol as anti-Leishmania treatment.

CASE PRESENTATION
A 1-year-old, entire, female crossbred dog with a history of unilateral nosebleed that occurred 4 months before referral.On physical examination, the dog was in good conditions, active and alert, normothermic and properly hydrated.Cardiac auscultation was within normal limits.Respiratory sounds were also normal and there was evidence of lymph node enlargement with generalised exfoliative dermatitis.
The dog was from the city of Zaragoza (41 • 39′ 24.6276″ N, 0 • 52′ 45.912″ W).), a very highly endemic area for canine leishmaniosis.The dog was not being treated with longacting topical anti-parasitic repellent against sandflies and/or mosquitoes.
Serum protein electrophoresis was also performed by agarose gel electrophoresis (AGE) system with HYDRAGEL Kit (SEBIA, Evry, France).Serum was electrophoresed for 21 minutes at 92 V hours and stained with diluted Amidoschwarz dye at pH 2 (4 g/L Amidoschwarz dye and 6.7% ethylene glycol).The AGE procedure was conducted according to the manufacturer's instructions, and commercial human serum was used as the control (normal control serum, Sebia, Evry, France).The electrophoretic curve for each sample was displayed.Protein fractions were determined as the percentage optical absorbance, and the absolute concentration in grams per decilitre was automatically calculated from the total serum protein concentration.Serum protein electrophoresis was run manually with agarose gels (Sebia, Evry, France) and densitometer (Shimadzu CS-9000, Kyoto, Japan) was used for scanning the electrophoretograms.
As canine leishmaniosis was the most prevalent disease with the presence of skin lesions, epistaxis and elevation of total proteins, it was necessary to rule out L. infantum performing a quantitative serology based on in-house ELISA technique. 8Briefly, each plate was coated lightly with 100 μl/well of the 20 μg/ml antigen solution (strain MHOM/FR/78/LEM 75 belonging to L. infantum zimodeme MON-1) in 0.1 M carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at 4 • C. Plates were then frozen and stored at −20 • C. One hundred microlitres of dog serum, diluted 1:800 in phosphate buffered saline containing 0.05% Tween 20 (PBST) and 1% dry skimmed milk (PBST-M) were added to each well.The plates were incubated for 1 hour at 37 • C in a moist chamber.After washing the plates three times with PBST for 3 minutes, followed by one wash with PBS for 1 minute, 100 μl of protein A conjugated to horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:20,000 in PBST-M was added to each well.The plates were incubated for 1 hour at 37 • C in a moist chamber, followed by washes with PBST and PBS as described above.The substrate solution (ortho-phenylene-diamine) and stable peroxide substrate buffer (Thermo Fisher Scientific, Waltham, MA, USA) were added (100 μl per well) and developed for 20 ± 5 minutes at room temperature in the dark.The reaction was terminated by adding 100 μl of 2.5 M H 2 SO 4 to each well.Absorbance

LEARNING POINTS/TAKE HOME MESSAGES
• To the best of our knowledge, this is the first case report describing the presence of an important reduction of anti-Leishmania antibodies after starting anti-Leishmania treatment.• Canine leishmaniosis should be included in the differential diagnostic list for patients presenting presumptive polyclonal spike in the beta fraction in endemic areas of Leishmania infantum infection.• C-reactive protein could be used as a biomarker in association with serum protein electrophoresis to evaluate the treatment response against Leishmania.• Low levels of immunoglobulin A deficiency could be detected in dogs with infectious conditions such Leishmania infantum.
values were read at 492 nm (reference wavelength) in an automatic microELISA reader (ELISA Reader Labsystems Multiskan, Midland, Canada).Each plate included serum samples from a dog infected with L. infantum as confirmed by cytological examination as a positive control (calibrator) and serum samples from a healthy, non-infected dog from the blood donor programme as a negative control.The same calibrator serum sample was used for all assays, and the plates with an interassay variation greater than 10% were tested again.All samples and controls were analysed in duplicate.The results were quantified as ELISA UNIT (EU) compared to a positive control serum sample used as a calibrator that was arbitrarily set to 100 EU.The cutoff value was set to 30 EU (mean + 4 standard deviations of values from 70 apparently healthy dogs from a non-endemic area and that were not included in this study).Sera with an EU ≥200 were classified as high positive, with an EU ≥100 and <200 as moderate positive, and with an EU >30 and <100 as low positive.All samples with an optical density (OD) equal or higher than 3 were studied using a twofold serial dilution. 9The samples were started at 1:800 dilution and continued for 10 further dilutions in the same ELISA plate.The results were quantified as EU related to a calibrator arbitrarily set at 100 EU with an OD of 1.00 at the 1:800 dilution.The values of the dilutions at which the OD was close to 1.00 was chosen for the calculation of the EU using the following formula: (sample OD/calibrator OD) × 100 × dilution factor.Laboratory findings revealed total protein increase, hyperglobulinemia, anaemia and CPR.The rest of the biochemical parameters were within the reference ranges.Urinalysis showed no abnormalities.Serum protein electrophoresis was also performed to investigate the nature of hyperglobulinemia.The electrophoresis revealed spike in the beta fraction (Figure 1).
Protein bands, forming the different electrophoretic regions, were identified using electrophoretic immunofixation (IFE) (Hydragel 4 IF Sebiat, Evry, France), a highly specific method was used to identify the exact location of proteins. 10This method is based on the recognition of a single protein by addition of a specific antibody (antisera) to the agarose gel substrate after electrophoretic migration (Hydrasys 2 Scan Focusing, Sebia, Evry.France).In our case, the antisera used was the same that has been applied in human medicine based on company recommendations due the conserved structure among mammals and the possibility of cross-reaction phenomenon among human and dog antibodies.No monoclonal band was observed in any of the three major immunoglobulins analysed, classifying it as polyclonal pattern composed by IgM and IgG and the absence of IgA (Figure 2).
Protein levels (IgG, IgA and IgM) were analysed in the serum samples by nephelometry.IgG, IgA and IgM were analysed using IMMAGE 800 (Beckman-Coulter, USA) nephelometer.Additionally, a total of 10 dogs, including different breeds with similar age to the present case report and classified as healthy, were included to evaluate the limit of detection of this technique.All dogs had higher levels than detection limit.

DIFFERENTIAL DIAGNOSIS
There are different causes of hyperglobulinemia in dogs. 10his alteration could be the result from increases in any or all the serum protein fractions, including α-globulins, β-globulins and γ-globulins.In the present case report, the shape of the electrophoretogram was very useful to characterise the hyperglobulinemia with the presence of a polyclonal betagammopathy that accompanies an elevation in γ-globulins as a consequence of L. infantum antigenic stimulation.This situation could be explained because immunoglobulins such as IgA, IgM and specific subclasses of IgG migrate in the β-globulin fraction.The most common causes of polyclonal gammopathy include inflammatory diseases (infectious, immune mediated), liver disease and suppurative dermatopathies.

TREATMENT
Treatment was focused on the parasite L. infantum.An anti-Leishmania therapeutic protocol was established with allopurinol at 10 mg/kg twice a day PO sine die and meglumine antimoniate at 40 mg/kg twice daily (BID) subcutaneously for 5 weeks.The patient did not require any other treatments.
An anti-Leishmania therapeutic protocol was established with allopurinol at 10 mg/kg BID PO sine die and meglumine antimoniate for 3 weeks at increasing doses every week to control possible drug adverse effects from 25 mg/kg BID the first week to 40 mg/kg BID the third week subcutaneously. 6,14

OUTCOME AND FOLLOW-UP
The nosebleed was resolved in 15 days after starting anti-Leishmania treatment, while ulcerative dermatitis was resolved 2 months later (Day 125).Follow-up visits were carried out during the period of meglumine antimoniate treatment.Four months after completing the meglumine antimoniate treatment, a follow-up visit to the attending veterinarian was made.At this follow-up visit, a physical examination and laboratory test were performed including CBC, serum biochemistry and serum protein electrophoresis (Table 1).In addition, serum protein electrophoresis using AGE was performed detecting a normal pattern serum protein electrophoresis (Figure 3) with the presence of IgG pattern detected by IFE (Figure 4).

DISCUSSION
Selective immunoglobulin A deficiency (IgAD) is the most prevalent type of primary immunodeficiency in humans and dogs. 15In humans, this selective immunodeficiency may be   asymptomatic or on the contrary, the presence of insidious symptoms from mild to severe, including respiratory infections, gastrointestinal infections, autoimmunity diseases and/or allergic diseases.It was first reported in the early 1960s, 16 and 20 years later, the presence of this selective IgAD was described in dogs. 17It is important to point out that IgA concentrations are found to vary widely between breeds, which explains the lack of a generally accepted diagnostic reference range for IgA deficiency in dogs. 18However, in the present case report, IFE technique determined the absence of IgA pattern before the treatment and after the treatment at Day 185 (Figure 4).Moreover, IgA levels were lower than detection limit of the second technique performed before and after the treatment (Table 1).Generally, canine immunodeficiency disorders are less characterised in comparison to human medicine.Two different types of canine immunodeficiencies are described: primary immunodeficiency associated to puppies and typically breed-related or secondary immunodeficiency in adults.This second condition in dogs is a consequence of a variety of causes, including drugs, neoplastic disorders, infectious diseases, inflammatory and allergic diseases among others.In humans and dogs, these disorders are associated with a mutation in the gene encoding the Ig alpha chain, with a relative low concentration in mucosal areas or in the serum.Selective IgAD has been described in several breeds, including German shepherd dog and English bull terrier. 19,20anine leishmaniosis is a vector borne disease caused by L. infantum.The parasite is able to compromise the dog's immune response, making it susceptible to other co-infections and/or disorders. 21oexistence of Leishmania and a selective IgAD is not described in the literature.It is difficult to establish the cause-effect relationship.If the IgAD is the consequence of the presence of the parasite or by contrast, the selective immunodeficiency may predispose an increase in the susceptibility to the infection.Some different factors, including breed, concomitant diseases (infectious diseases, neoplasia, endocrinopathies), immunosuppression status and nutritional factors, can directly influence the susceptibility to infection. 22Although it is not possible to explain the relationship between the parasitic disease and selective immunoglobulin deficiency, the results of this case report document the ability of Leishmania to infect dogs with a type of immunodeficiency.In the present case report, selective IgAD is not secondary to the parasite infection because 6 months after clinical presentation, no clinical signs or clinicopathological abnormalities have been detected except for the fact that the dog presents this selective IgAD.
The relationship between the presence of anti-Leishmania IgA and dogs suffering clinical leishmaniosis has been evaluated in urine and serum samples. 23The role of the IgA is associated to mucosal immunology as first level of protection in mucosal secretions.The absence of IgA in serum samples might favour the presence of the parasite in mucosal areas, including nasal cavity, with the appearance of lesions such as epistaxis.
Traditionally, quantitative serology is not recommended before 6 months after initial anti-Leishmania treatment.However, recent evidences confirm that an early reduction of anti-Leishmania antibodies is detected after 30 days after the first meglumine antimoniate injection.In our case report, a marked reduction of the anti-Leishmania antibodies was detected during the meglumine administration based on the serial dilution method ELISA in comparison to one dilution method. 8The reason for this difference between one dilution and the ELISA serial dilution method is due to in the first case, a high anti-Leishmania antibodies concentration is present in the serum sample analysed.This condition produces a saturated signal in the ELISA technique, whereas the use of sample dilutions by serial dilution ELISA method decreases the amount of anti-Leishmania antibodies avoiding ELISA limits.The reduction of the level of anti-Leishmania antibodies during the meglumine antimoniate administration correlates with the clinical course and laboratory findings such as CPR.
The use of CPR as biomarker in canine leishmaniosis has been evaluated in dogs undergoing leishmaniosis treatment. 24he measurement of this APP could be of interest because it could help clinicians to make decisions during the followup.A decrease in serum protein electrophoresis during meglumine antimoniate treatment is detected even before the improvement.The reduction of CPR concentration is a laboratory parameter that can help clinicians to evaluate the response to anti-Leishmania therapy in short-term, together with the quantitative serology.
The clinician should be aware that early reduction of anti-Leishmania antibodies can be detected using serial dilution ELISA method after starting meglumine antimoniate treatment in dogs with leishmaniosis.Moreover, anti-Leishmania antibodies reduction correlates with CPR reduction during meglumine antimoniate administration.

F
I G U R E  Electrophoretogram using agarose gel electrophoresis.A presumptive polyclonal spike in the beta fraction is detected F I G U R E  Protein characterisation using immunofixation before the treatment.A polyclonal pattern composed by IgM and IgG is detected

F
I G U R E  Electrophoretogram using agarose gel electrophoresis at Day 185.The electrophoresis revealed normal pattern serum protein F I G U R E  Protein characterisation using immunofixation after treatment at Day 185.An IgG pattern is detected Haematological, biochemical parameters and serology determined in the leishmaniotic dog at the first veterinary examination before treatment and during the follow-up TA B L E  Abnormalities are highlighted in bold.Abbreviations: A/G, albumin:globulin ratio; Ig, immunoglobulin; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; NA, not available; RBC, red blood count; RDW, red blood cell distribution; WBC, white blood count.