**Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes**

Natasa Tozon, Mauro Pistello and Alessandro Poli

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51534

#### **1. Introduction**

374 Immunodeficiency

patterns*. J Virol.* 68(4):2230-8.

*Virol.* 80(7):3386-94a.

80(15):7744-7b.

comparison of the FIV and human immunodeficiency virus type 1 evolutionary

Willett, B. J., McMonagle, E. L., Ridha, S., Hosie, M. J. (2006). Differential utilization of CD134 as a functional receptor by diverse strains of feline immunodeficiency virus. *J.* 

Willett, B. J., McMonagle, E. L., Bonci, F., Pistello, M. & Hosie, M. J. (2006). Mapping the domains of CD134 as a functional receptor for feline immunodeficiency virus. *J. Virol.*

Wolf-Jäckel, G. A., Cattori, V., Geret, C. P., Novacco, M., Meli, M. L., Riond, B., Boretti, F. S., Lutz, H., Hofmann-Lehmann, R. (2012). Quantification of the humoral immune response and hemoplasma blood and tissue loads in cats coinfected with 'Candidatus Mycoplasma haemominutum' and feline leukemia virus. *Microb. Pathog.* 53(2):74-80. Yuan, X., Salama, A. D., Dong, V., Schmitt, I., Najafian, N., Chandraker, A., Akiba, H., Yagita, H. & Sayegh, M. H. (2003). The role of the CD134-CD134 ligand costimulatory

pathway in alloimmune responses in vivo. *J. Immunol.* 170(6):2949-55.

The feline immunodeficiency virus (FIV) is a lentivirus isolated from domestic cats with an acquired immunodeficiency syndrome-like condition, named feline AIDS (F-AIDS). The major immunological abnormalities observed in FIV-infected cats included a profound decline in the absolute number of the CD4+ T cells that caused the inversion of the CD4+/CD8+ T cell ratio and increased susceptibility to opportunistic infections and various clinic-pathological conditions [1]. FIV viruses encompass a large group of strains classified in subgroups from A to E, which are unevenly distributed geographically and have an intersubtype diversity > 26% [2]. The isolates used in our study were Petaluma, of group A, and Pisa-M2, a local isolate belonging to group B, which encloses all isolates hitherto sequenced and circulating in Italy [3 4]. Serological screenings performed in the past demonstrated that the virus is distributed worldwide and incidence varies from 1 – 14% in healthy cats and up to 44% in sick cats. As other lentiviruses, FIV is a complex retrovirus with structural genes *gag, pol* and *env*, and a few accessory genes [5]; *gag* encodes the capsid protein p24, used in most diagnostic tests, and other inner structural proteins, *pol* encodes the enzymes necessary for viral replication and therefore targeted by most anti-lentiviral drugs, and *env* encode the outer glycoprotein (gp95) and trans-membrane protein (gp36) serving as viral receptor and, being constantly under immunological pressure, the less conserved proteins among the different subtypes. Like the human immunodeficiency virus (HIV), the gp95 is comprised of variable and conserved regions and binds the CD134 molecule, the FIV primary receptor [6]. Studies on HIV have shown that some conserved epitopes are accessible for neutralizing antibodies, while the co-receptor binding site is composed by interspersed domains. The binding site remains largely hidden and is therefore inaccessible for mentioned antibody. Whereas overall HIV and FIV Env structure is maintained [7], HIV uses various coreceptors, including a range transmembrane domain G-protein-coupled receptors. In

© 2012 Tozon et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

contrast,, all FIV strains tested so far use CXCR4 as a co-receptor [7]. Cats, once infected with FIV, remain infected life-long and in the face of strong humoral and cell-mediated immune responses that appear shortly after the initial viremic phase [2].

Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes 377

Animal Pathology of the University of Pisa by veterinarian practitioners and owners from different part of Tuscany, Italy. All the subjects were submitted to an accurate clinical examination and classified according to a previously proposed classification [18]. Thirty-six of these subjects were sacrificed and immediately necropsied. There was no investigator bias in this sampling as the only criterion used was the owner's consent to post-mortem examination. Fifteen cats who tested positive for leukemia virus p27 antigen (CiteCombo FIV\_FeLV, Agritech Systems, Portland, Me, USA) and feline infectious peritonitis antibodies (Diasystems Celisa FIP, Tech America, Omaha, NE, USA) were excluded from the study, as these viruses are known to be associated with renal alterations. The 21 selected cats were 16 males and 5 females, with a mean age of 96.0 months ± 40.3 months, 10 cats were in the

Fifty-six specific pathogen free SPF cats, infected with Petaluma and/or Pisa-M2 isolates at six-ten months of age, were included in the study. All subjects were females that at time of analyses aged between 2 and 6 years (mean age of 47.8 months ± 14.4 months). Seven-teen animals were inoculated with Petaluma (group 1), 28 with Pisa-M2 isolate (group 2), and 11 were first inoculated with Petaluma and, one year later, superinfected with Pisa-M2 (group 3). Petaluma isolate was obtained from supernatant of persistently infected FL4 cells [19], while Pisa-M2 was a local isolate propagated in vivo by monthly passages in SPF cats and never passaged in vitro [20]. Animals were inoculated intravenously with either 2 ml of freshly collected blood (Pisa-M2) or 20 cat infectious dose 50% of FL-4 supernatant (Petaluma). All cats seroconverted in 4-6 weeks and were characterized for a steady reduction in the number of circulating CD4+ lymphocyte that approximately halved in 1 year. Four FIV-negative,SPF cats were used as negative controls (group C). Infected and control cats were housed in biosafety hazard level 3 conditions at the Retrovirus Center of the University of Pisa, and were daily monitored for clinical conditions throughout the observation period. Physical examination was performed weekly for the first two months postinfection (pi) and then monthly. At 12, 24, 30, 36 and 48 months pi, randomly selected

Urine specimens were obtained by cystocentesis. After centrifugation, supernatants were used to determine protein and creatinine concentration using two commercial assays (BioRad, Richmond, Calif., USA, and Creatinine - Jaffe method, Verbena, Milano, Italy, respectively). In cats with marked proteinuria (>2 g/L), urine protein/urine creatinin ratio (UPC) was calculated using the following formula: P(g/L) x 100 / (Cr mmol/L / 0.0885). Protein qualitative analysis was performed with sodium dodecyl sulfate-polyacrylamide gel

Blood samples for determination of biochemical profile were collected into serum separator tubes (Vacuette, Greiner Bio-One, Kremsmunster, Austria) and stood for 30 min at 4°C to clot, then centrifuged (1300 g for 10 min) to separate the serum. Serum samples were assayed for selected biochemical parameters including urea, creatinine, total protein,

symptomatic phase of infection phase, while 11 had full-blown F-AIDS.

animals were deeply anesthesized, and euthanized for necropsy.

**2.2. Biochemistry and urine analysis** 

(SDS-PAGE) according to Leamlli [21].

The acute phase of infection lasts a few days to a few weeks and is asymptomatic in a large proportion of cats. If clinically overt, it manifests with fever, lethargy and peripheral lymphadenomegaly with possible neutropenia. The acute phase eventually subsides and the infected cat enters in asymptomatic period that typically lasts 4 to 6 years or is life-long in some cats. In 30% cats and with percentages that greatly depend on cofactors and cat lifestyle [2], the infection proceeds to the last stage, the F-AIDS, that is characterized by profound immunodeficiency and, consequently, the presence of secondary infections sustained by viruses, bacteria, fungi, or protozoa, and various neoplastic diseases. Like HIV, FIV also infects and may damage the central nervous system as demonstrated in the past in experimental conditions [8-11]. As mentioned, clinical presentation and outcome of disease depend upon a combination of secondary factors and host immune responses. Immunodeficiency combined with immunostimulation by various factors most frequently results in the emergence of severe forms of gingivostomatitis, chronic rhinitis, lymphadenopathy, weight loss and immune-mediated glomerulonephritis [2]. Weaver and co-workers reported reproductive failure in FIV-infected cats. Viral DNA in placental and fetal tissues in affected cats was confirmed by PCR [12].

Despite detailed knowledge of most clinicopathological features during FIV infection, information on renal involvement is limited. Unspecified renal abnormalities were reported in some infected cats living in Australia [13] and in 5.5% of those living in New Zealand [14]. Ishida et al found that 9.3% of 700 Japanese FIV-infected cats presented clinical signs of renal diseases [15]. Most pathological findings observed in the kidney of naturally FIVinfected cats resemble those described in HIV-infected patients [16], but it is not clear whether FIV has a direct role in the induction of the renal damage or accelerates a phenomenon triggered by other factors. The fact that renal damage is mostly found in natural FIV infection supports the latter hypothesis [17]. However, since there are no detailed descriptions of the renal lesions found in experimentally FIV-infected cats this issue is still open. The aim of our study was to investigate the histological renal alterations caused by FIV in animals experimentally inoculated with FIV strains of different pathogenicity and at different times post-infection. Here, specific pathogen free (SPF) cats singly or doubly infected with Petaluma and Pisa-M2 were housed in germ-free conditions to exclude the influence of other pathogens. The pathological findings in these animals were compared with those found in naturally infected cats. These results were also compared to those found in HIV patients.

#### **2. Material and methods**

#### **2.1. Cats**

Ninety-nine naturally infected cats were collected from 1990 to 1993 and after diagnosis of FIV infection performed by western blot. Animals were referred to the Department of Animal Pathology of the University of Pisa by veterinarian practitioners and owners from different part of Tuscany, Italy. All the subjects were submitted to an accurate clinical examination and classified according to a previously proposed classification [18]. Thirty-six of these subjects were sacrificed and immediately necropsied. There was no investigator bias in this sampling as the only criterion used was the owner's consent to post-mortem examination. Fifteen cats who tested positive for leukemia virus p27 antigen (CiteCombo FIV\_FeLV, Agritech Systems, Portland, Me, USA) and feline infectious peritonitis antibodies (Diasystems Celisa FIP, Tech America, Omaha, NE, USA) were excluded from the study, as these viruses are known to be associated with renal alterations. The 21 selected cats were 16 males and 5 females, with a mean age of 96.0 months ± 40.3 months, 10 cats were in the symptomatic phase of infection phase, while 11 had full-blown F-AIDS.

Fifty-six specific pathogen free SPF cats, infected with Petaluma and/or Pisa-M2 isolates at six-ten months of age, were included in the study. All subjects were females that at time of analyses aged between 2 and 6 years (mean age of 47.8 months ± 14.4 months). Seven-teen animals were inoculated with Petaluma (group 1), 28 with Pisa-M2 isolate (group 2), and 11 were first inoculated with Petaluma and, one year later, superinfected with Pisa-M2 (group 3). Petaluma isolate was obtained from supernatant of persistently infected FL4 cells [19], while Pisa-M2 was a local isolate propagated in vivo by monthly passages in SPF cats and never passaged in vitro [20]. Animals were inoculated intravenously with either 2 ml of freshly collected blood (Pisa-M2) or 20 cat infectious dose 50% of FL-4 supernatant (Petaluma). All cats seroconverted in 4-6 weeks and were characterized for a steady reduction in the number of circulating CD4+ lymphocyte that approximately halved in 1 year. Four FIV-negative,SPF cats were used as negative controls (group C). Infected and control cats were housed in biosafety hazard level 3 conditions at the Retrovirus Center of the University of Pisa, and were daily monitored for clinical conditions throughout the observation period. Physical examination was performed weekly for the first two months postinfection (pi) and then monthly. At 12, 24, 30, 36 and 48 months pi, randomly selected animals were deeply anesthesized, and euthanized for necropsy.

#### **2.2. Biochemistry and urine analysis**

376 Immunodeficiency

contrast,, all FIV strains tested so far use CXCR4 as a co-receptor [7]. Cats, once infected with FIV, remain infected life-long and in the face of strong humoral and cell-mediated immune

The acute phase of infection lasts a few days to a few weeks and is asymptomatic in a large proportion of cats. If clinically overt, it manifests with fever, lethargy and peripheral lymphadenomegaly with possible neutropenia. The acute phase eventually subsides and the infected cat enters in asymptomatic period that typically lasts 4 to 6 years or is life-long in some cats. In 30% cats and with percentages that greatly depend on cofactors and cat lifestyle [2], the infection proceeds to the last stage, the F-AIDS, that is characterized by profound immunodeficiency and, consequently, the presence of secondary infections sustained by viruses, bacteria, fungi, or protozoa, and various neoplastic diseases. Like HIV, FIV also infects and may damage the central nervous system as demonstrated in the past in experimental conditions [8-11]. As mentioned, clinical presentation and outcome of disease depend upon a combination of secondary factors and host immune responses. Immunodeficiency combined with immunostimulation by various factors most frequently results in the emergence of severe forms of gingivostomatitis, chronic rhinitis, lymphadenopathy, weight loss and immune-mediated glomerulonephritis [2]. Weaver and co-workers reported reproductive failure in FIV-infected cats. Viral DNA in placental and

Despite detailed knowledge of most clinicopathological features during FIV infection, information on renal involvement is limited. Unspecified renal abnormalities were reported in some infected cats living in Australia [13] and in 5.5% of those living in New Zealand [14]. Ishida et al found that 9.3% of 700 Japanese FIV-infected cats presented clinical signs of renal diseases [15]. Most pathological findings observed in the kidney of naturally FIVinfected cats resemble those described in HIV-infected patients [16], but it is not clear whether FIV has a direct role in the induction of the renal damage or accelerates a phenomenon triggered by other factors. The fact that renal damage is mostly found in natural FIV infection supports the latter hypothesis [17]. However, since there are no detailed descriptions of the renal lesions found in experimentally FIV-infected cats this issue is still open. The aim of our study was to investigate the histological renal alterations caused by FIV in animals experimentally inoculated with FIV strains of different pathogenicity and at different times post-infection. Here, specific pathogen free (SPF) cats singly or doubly infected with Petaluma and Pisa-M2 were housed in germ-free conditions to exclude the influence of other pathogens. The pathological findings in these animals were compared with those found in naturally infected cats. These results were also compared to

Ninety-nine naturally infected cats were collected from 1990 to 1993 and after diagnosis of FIV infection performed by western blot. Animals were referred to the Department of

responses that appear shortly after the initial viremic phase [2].

fetal tissues in affected cats was confirmed by PCR [12].

those found in HIV patients.

**2. Material and methods** 

**2.1. Cats** 

Urine specimens were obtained by cystocentesis. After centrifugation, supernatants were used to determine protein and creatinine concentration using two commercial assays (BioRad, Richmond, Calif., USA, and Creatinine - Jaffe method, Verbena, Milano, Italy, respectively). In cats with marked proteinuria (>2 g/L), urine protein/urine creatinin ratio (UPC) was calculated using the following formula: P(g/L) x 100 / (Cr mmol/L / 0.0885). Protein qualitative analysis was performed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) according to Leamlli [21].

Blood samples for determination of biochemical profile were collected into serum separator tubes (Vacuette, Greiner Bio-One, Kremsmunster, Austria) and stood for 30 min at 4°C to clot, then centrifuged (1300 g for 10 min) to separate the serum. Serum samples were assayed for selected biochemical parameters including urea, creatinine, total protein,

albumin, using an automated analysis on a spectrophotometer (LKB Biochrom Ltd., Cambridge, UK). Serum albumin/globulin ratio was calculated.

Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes 379

periodic-acidic Schiff and Jones periodic acid-silver methenamine. Sections with inflammatory lesions were stained with Ziehl-Neelsen acid-fast and Gram to exclude bacterial infections. Amyloid was demonstrated by the alkaline Congo red staining with polarization on 8 μm sections [24]. Differentiation between primary and secondary amyloidosis was based on staining by a modified Romhanyi method with pre treatment with potassium permanganate [25] and on immunohistochemistry (IHC) with antibodies

The localization of IgG, IgA, IgM and C3 deposits was investigated by both indirect immunofluorescence method (IF) and streptavidin-biotin peroxidase method. IF was performed using as primary antibodies primary sheep monospecific antibodies to cat IgG, IgM, IgA and C3 (binding Site, Birmingham, UK) and a rabbit fluorescein anti-sheep IgG (vector Laboratorie, Burlingame, CA, USA), as previously described [16]. Control sections were incubated with normal sheep serum (Dako, Golstrup, Denmark) before treatment with the secondary antiserum. For IHC, sections were de-waxed in xylene, passed through a graded series of alcohols, and rehydrated in deionised water. For Ig and C3 localization, the tissues were digested with 0.5% protease (Protease XXIV; Sigma, Saint Louis, Mo, USA) in 0.05 M Tris-Hcl, pH 7.6. Endogenous peroxidases were exhausted with 0.5% hydrogen peroxide for 30 minutes and after that, three washes were performed in 0.05% Tween Tris Buffered Saline solution (TBST) at pH 7.6. Normal serum from the host species of the secondary antibody diluted 1/10 in TBST was added to the sections and incubated for 30 minutes at room temperature. After three washes, the primary antibodies diluted in TBST were applied and incubated for 1 hour at RT. The antisera used included unlabeled sheep anti cat IgG, IgA, IgM and C3, an anti-AA (murine monoclonal mc4 against human AA protein, culture supernatant and anti-cat AA and AL (polyclonal antibody of rabbit origin; kind gift of R. P. Link, University of Munich, Germany). After three washes, secondary biotinylated antibody (Vectastain®, Vector Labs Inc., Burlingame, CA, USA) was added and incubated for 30 minutes at RT. Peroxidase reaction was developed for 10 minutes using diaminobenzidine (DAB) (Impact DAB® ,Vector Labs inc., Burlingame, CA, USA) and blocked with deionised water. Negative controls were performed omitting the primary antibody and replacing the antibody with normal sheep or rabbit serum or murine subclass matched (IgG1) unrelated primary monoclonal

Statistical analysis was performed using the statistical package SPSS Advanced Statistics 13.0 (SPSS Inc., Chicago, IL, USA). Chi-square test was used to investigate the significance of the relationship between protein expression and individual variables. Statistical significance

against amyloid A protein.

antibody.

**2.8. Statistics** 

was based on a 5% (0.05) significance level.

**2.7. Immunohistochemistry** 

#### **2.3. FIV serology**

All cats were screened for FIV antibodies and feline leukaemia virus antigen by a commercial ELISA (CITE COMBO FIV-FeLV;Agritech Systems, Portland, Maine) according to manufacturer's instructions.

FIV antibodies, detected with above rapid test, were confirmed by Western blot (WB). FIV antigen for WB analysis was produced from persistently FIV-infected FL4 T-cells. The virus was pelleted from tissue cultures medium, purified throughout a 10 to 50% continuous sucrose gradient and then disrupted by sonication followed by treatment with Triton X-100. Viral proteins were separated by electrophoresis with 12% SDS-PAGE and blotted onto nitrocellulose by standard methods. The nitrocellulose sheets were then blocked with 1% bovine serum albumin, cut into 0.5 cm wide strips, dried, and stored in the cold until use. Individual strips were incubated for two hours with serum samples diluted 1:100 in PBS-Tween 20 (0.05%), washed thoroughly, incubated with horseradish peroxidase-conjugated rabbit anti-cat immunoglobulin G (Bethyl Laboratories, Montgomery, Texas) for one hour. The strips were washed and incubated with 0.05% diaminobenzidine and 0.01% of H O (what is this?) in 0.1M Tris pH 7.4 to visualize antigen-antibody binding. Strips were read by densitometric scanning (BioRad) immediately after the reaction was stopped with distilled water. The molecular weights of the reactive proteins were established by comparison with prestained low-molecular-weight markers (BioRad). Each WB contained positive and negative control sera. The sample was scored positively when at least two of p25, p31, gp40, gp65, and gp95 FIV-specific bands were clearly detectable.

#### **2.4. PCR**

Buffy coat of 2 ml whole blood samples was used to detect FIV DNA. FIV DNA extraction and amplification was performed as described [22]. All precautions were taken to avoid possible contamination and samples were examined at least twice in separate experiments. DNA from peripheral blood mononuclear cells (PBMC) of uninfected cats and from Petaluma-infected FL-4 cells were used as negative and positive controls, respectively.

#### **2.5. Flow citometry**

T lymphocyte subsets were examined by flow cytometric analysis as described [23]. Flow cytometry analysis was performed by using fluorescein conjugated murine monoclonal antibodies to feline CD4 and CD8 T-cells surface markers (Southern, Biotech, Birmingham, AL,USA) and an Epics Elite cell analyzer (Coulter Electronics, Hialeah, Fla.).

#### **2.6. Histology**

Renal tissue samples were fixed in 10% buffered formalin solution and embedded in paraffin. 3μm thick sections from each specimen were stained with hematoxylin and eosin, periodic-acidic Schiff and Jones periodic acid-silver methenamine. Sections with inflammatory lesions were stained with Ziehl-Neelsen acid-fast and Gram to exclude bacterial infections. Amyloid was demonstrated by the alkaline Congo red staining with polarization on 8 μm sections [24]. Differentiation between primary and secondary amyloidosis was based on staining by a modified Romhanyi method with pre treatment with potassium permanganate [25] and on immunohistochemistry (IHC) with antibodies against amyloid A protein.

#### **2.7. Immunohistochemistry**

378 Immunodeficiency

**2.4. PCR** 

**2.5. Flow citometry** 

**2.6. Histology** 

**2.3. FIV serology** 

to manufacturer's instructions.

albumin, using an automated analysis on a spectrophotometer (LKB Biochrom Ltd.,

All cats were screened for FIV antibodies and feline leukaemia virus antigen by a commercial ELISA (CITE COMBO FIV-FeLV;Agritech Systems, Portland, Maine) according

FIV antibodies, detected with above rapid test, were confirmed by Western blot (WB). FIV antigen for WB analysis was produced from persistently FIV-infected FL4 T-cells. The virus was pelleted from tissue cultures medium, purified throughout a 10 to 50% continuous sucrose gradient and then disrupted by sonication followed by treatment with Triton X-100. Viral proteins were separated by electrophoresis with 12% SDS-PAGE and blotted onto nitrocellulose by standard methods. The nitrocellulose sheets were then blocked with 1% bovine serum albumin, cut into 0.5 cm wide strips, dried, and stored in the cold until use. Individual strips were incubated for two hours with serum samples diluted 1:100 in PBS-Tween 20 (0.05%), washed thoroughly, incubated with horseradish peroxidase-conjugated rabbit anti-cat immunoglobulin G (Bethyl Laboratories, Montgomery, Texas) for one hour. The strips were washed and incubated with 0.05% diaminobenzidine and 0.01% of H O (what is this?) in 0.1M Tris pH 7.4 to visualize antigen-antibody binding. Strips were read by densitometric scanning (BioRad) immediately after the reaction was stopped with distilled water. The molecular weights of the reactive proteins were established by comparison with prestained low-molecular-weight markers (BioRad). Each WB contained positive and negative control sera. The sample was scored positively when at least two of p25, p31, gp40,

Buffy coat of 2 ml whole blood samples was used to detect FIV DNA. FIV DNA extraction and amplification was performed as described [22]. All precautions were taken to avoid possible contamination and samples were examined at least twice in separate experiments. DNA from peripheral blood mononuclear cells (PBMC) of uninfected cats and from Petaluma-infected FL-4 cells were used as negative and positive controls, respectively.

T lymphocyte subsets were examined by flow cytometric analysis as described [23]. Flow cytometry analysis was performed by using fluorescein conjugated murine monoclonal antibodies to feline CD4 and CD8 T-cells surface markers (Southern, Biotech, Birmingham,

Renal tissue samples were fixed in 10% buffered formalin solution and embedded in paraffin. 3μm thick sections from each specimen were stained with hematoxylin and eosin,

AL,USA) and an Epics Elite cell analyzer (Coulter Electronics, Hialeah, Fla.).

Cambridge, UK). Serum albumin/globulin ratio was calculated.

gp65, and gp95 FIV-specific bands were clearly detectable.

The localization of IgG, IgA, IgM and C3 deposits was investigated by both indirect immunofluorescence method (IF) and streptavidin-biotin peroxidase method. IF was performed using as primary antibodies primary sheep monospecific antibodies to cat IgG, IgM, IgA and C3 (binding Site, Birmingham, UK) and a rabbit fluorescein anti-sheep IgG (vector Laboratorie, Burlingame, CA, USA), as previously described [16]. Control sections were incubated with normal sheep serum (Dako, Golstrup, Denmark) before treatment with the secondary antiserum. For IHC, sections were de-waxed in xylene, passed through a graded series of alcohols, and rehydrated in deionised water. For Ig and C3 localization, the tissues were digested with 0.5% protease (Protease XXIV; Sigma, Saint Louis, Mo, USA) in 0.05 M Tris-Hcl, pH 7.6. Endogenous peroxidases were exhausted with 0.5% hydrogen peroxide for 30 minutes and after that, three washes were performed in 0.05% Tween Tris Buffered Saline solution (TBST) at pH 7.6. Normal serum from the host species of the secondary antibody diluted 1/10 in TBST was added to the sections and incubated for 30 minutes at room temperature. After three washes, the primary antibodies diluted in TBST were applied and incubated for 1 hour at RT. The antisera used included unlabeled sheep anti cat IgG, IgA, IgM and C3, an anti-AA (murine monoclonal mc4 against human AA protein, culture supernatant and anti-cat AA and AL (polyclonal antibody of rabbit origin; kind gift of R. P. Link, University of Munich, Germany). After three washes, secondary biotinylated antibody (Vectastain®, Vector Labs Inc., Burlingame, CA, USA) was added and incubated for 30 minutes at RT. Peroxidase reaction was developed for 10 minutes using diaminobenzidine (DAB) (Impact DAB® ,Vector Labs inc., Burlingame, CA, USA) and blocked with deionised water. Negative controls were performed omitting the primary antibody and replacing the antibody with normal sheep or rabbit serum or murine subclass matched (IgG1) unrelated primary monoclonal antibody.

#### **2.8. Statistics**

Statistical analysis was performed using the statistical package SPSS Advanced Statistics 13.0 (SPSS Inc., Chicago, IL, USA). Chi-square test was used to investigate the significance of the relationship between protein expression and individual variables. Statistical significance was based on a 5% (0.05) significance level.
