State of the art: FIP and FIV diagnostics Saverio Paltrinieri, DVM, PhD, dipl ECVCP Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Italy Feline Infectious Peritonitis (FIP) is due to a feline coronavirus (FCoV). The FIP Virus (FIPV) arise from the widely diffused and low pathogenic Feline Enteric Coronavirus (FECV) by a series of mutation. The two viruses cannot be distinguished by serology or PCR tests. Nevertheless a PCR test able to identify replicating FCoV within macrophages has recently been proposed. When this test will be available, the FIPV might be differentiated from the FECV. Unlike FECV, the FIPV is able to replicate within macrophages, which distributed it throughout the body. The immune reaction of the host determines the appearance of granulomatous lesions (dry FIP) or of a diffuse vasculitis which induces cavitary effusions (wet form). The two main diagnostic challenges that can be encountered in practice are the diagnosis of the disease and the detection of FCoV carriers. Due to the above mentioned difficulties in differentiating FECV and FIPV, antibodies and FCoV genome can be detected also in cats with symptoms consistent with FIP but affected by diseases other than FIP: the diagnosis of FIP must thus be based on surrogate markers of the diseases. Haematology can be used to detect non regenerative normocytic normochromic anemia, neutrophilia and lymphopenia. The latter finding is highly suggestive of FIP and flow cytometric analysis of circulating lymphocytes shows that all the lymphocyte subsets decrease during FIP: this signs is highly non-specific, but flow cytometry might allow to rule out the suspicion of FIP in the case of normal B or CD8 counts. Elevated globulin levels with hypoalbuminemia and increased 2- and -globulins are typical electrophoretic patterns of cats with FIP. Among serum proteins, Acute Phase Proteins (APP) such as Serum Amyloid A (SAA) and 1-acid glycoprotein (AGP) strongly increase during FIP. In particular, AGP increases are highly diagnostic for FIP: moreover, in cats with FIP AGP is hyposyalilated, but the diagnostic significance of this finding should be still verified. In the case of suspected wet FIP, the analysis of the effusions is the best diagnostic method. Specifically, effusions collected during FIP have a high protein and globulin content, high AGP levels, and a cytological pattern consistent with a non-specific inflammatory process characterised by a proteinaceous basophilic background. The immunocytochemical detection of FCoVs within the macrophages detectable in cytocentrifuged effusion can further confirm the suspicion of FIP. The detection of histologic lesions consistent with FIP is considered the only conclusive test for FIP, especially in the case immunohistochemical stains for FCoVs are positive. Citology and histology consistent with FIP, or FCoV-positive macrophages can also be found “in vivo” in ultrasound-guided tru-cut biopsies (TCB) or on fine needle aspiration biopsies (FNA) from liver and kidney. The identification of FCoV carriers is mainly based on serology or on the detection of FCoV RNA by repeated PCR on fecal samples: transient, pesistent or recurrent sheddders can thus be identified. The future of FIP research is the diagnosis of susceptible animals: some clinico-pathological parameters (lymphocyte subset counts, APP levels, degree of AGP syalilation, cytokine levels) are strongly different in FCoV positive cats that do not develop the disease compared to cats with FIP. Feline immunodeficiency virus (FIV) is a retrovirus that is transmitted among cat population by feline bites, due to its presence on the saliva of infected cats. After bites, the virus persistently infect CD4 lymphocytes, where it is detectable as integrated proviral DNA 2-4 weeks post-infection (p.i.). During this Acute Phase (AP), non-specific symptoms (fever, neutropenia, anemia) can be detected. The viremia decrease to very low levels 2-4 months p.i., when circulating antibodies appear. This lead to the Asymptomatic Carrier (AC) state, lasting for several years, during which a Persistent Generalised Lymphadenopathy (PGL) might appear. In terminal stages, the lymphocyte infection lead to a progressive immunodepression (AIDS Related Complex or ARC) with the appearance of opportunistic infection such as skin lesions, stomatitis-gingivitis or of haematological disorders and to a final collapse of immune system (Feline AIDS or FAIDS). From the above reported brief description of the course of the disease arise that symptoms are non-specific and the diagnosis should be based on the detection of circulating antibodies or of proviral DNA. Antibodies can be detected during AC, PGL and, occasionally, ARC phases. Several in-house ELISA or immunochromatografic kits can be used to detect antibodies: doubt results should be verified by IFA or Western Blotting (gold standard). Proviral DNA can be detected by PCR since 2 weeks p.i. until the death. Real Time or TaqMan PCR might allow us to quantify the viral burden in order to monitor the progression of the disease. Similarly, the disease can be staged by measuring with a flow cytometer the CD4/CD8 lymphocyte ratio: CD4 lymphocytes, in fact, progressively decrease during the diseases, while CD8 lymphocytes decreases only in terminal stages.

State of the art: FIP and FIV diagnostics / S. Paltrinieri. ((Intervento presentato al 2. convegno 2nd CE day of the ESVCP-ECVCP tenutosi a Uppsala nel 2003.

State of the art: FIP and FIV diagnostics

S. Paltrinieri
Primo
2003

Abstract

State of the art: FIP and FIV diagnostics Saverio Paltrinieri, DVM, PhD, dipl ECVCP Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Italy Feline Infectious Peritonitis (FIP) is due to a feline coronavirus (FCoV). The FIP Virus (FIPV) arise from the widely diffused and low pathogenic Feline Enteric Coronavirus (FECV) by a series of mutation. The two viruses cannot be distinguished by serology or PCR tests. Nevertheless a PCR test able to identify replicating FCoV within macrophages has recently been proposed. When this test will be available, the FIPV might be differentiated from the FECV. Unlike FECV, the FIPV is able to replicate within macrophages, which distributed it throughout the body. The immune reaction of the host determines the appearance of granulomatous lesions (dry FIP) or of a diffuse vasculitis which induces cavitary effusions (wet form). The two main diagnostic challenges that can be encountered in practice are the diagnosis of the disease and the detection of FCoV carriers. Due to the above mentioned difficulties in differentiating FECV and FIPV, antibodies and FCoV genome can be detected also in cats with symptoms consistent with FIP but affected by diseases other than FIP: the diagnosis of FIP must thus be based on surrogate markers of the diseases. Haematology can be used to detect non regenerative normocytic normochromic anemia, neutrophilia and lymphopenia. The latter finding is highly suggestive of FIP and flow cytometric analysis of circulating lymphocytes shows that all the lymphocyte subsets decrease during FIP: this signs is highly non-specific, but flow cytometry might allow to rule out the suspicion of FIP in the case of normal B or CD8 counts. Elevated globulin levels with hypoalbuminemia and increased 2- and -globulins are typical electrophoretic patterns of cats with FIP. Among serum proteins, Acute Phase Proteins (APP) such as Serum Amyloid A (SAA) and 1-acid glycoprotein (AGP) strongly increase during FIP. In particular, AGP increases are highly diagnostic for FIP: moreover, in cats with FIP AGP is hyposyalilated, but the diagnostic significance of this finding should be still verified. In the case of suspected wet FIP, the analysis of the effusions is the best diagnostic method. Specifically, effusions collected during FIP have a high protein and globulin content, high AGP levels, and a cytological pattern consistent with a non-specific inflammatory process characterised by a proteinaceous basophilic background. The immunocytochemical detection of FCoVs within the macrophages detectable in cytocentrifuged effusion can further confirm the suspicion of FIP. The detection of histologic lesions consistent with FIP is considered the only conclusive test for FIP, especially in the case immunohistochemical stains for FCoVs are positive. Citology and histology consistent with FIP, or FCoV-positive macrophages can also be found “in vivo” in ultrasound-guided tru-cut biopsies (TCB) or on fine needle aspiration biopsies (FNA) from liver and kidney. The identification of FCoV carriers is mainly based on serology or on the detection of FCoV RNA by repeated PCR on fecal samples: transient, pesistent or recurrent sheddders can thus be identified. The future of FIP research is the diagnosis of susceptible animals: some clinico-pathological parameters (lymphocyte subset counts, APP levels, degree of AGP syalilation, cytokine levels) are strongly different in FCoV positive cats that do not develop the disease compared to cats with FIP. Feline immunodeficiency virus (FIV) is a retrovirus that is transmitted among cat population by feline bites, due to its presence on the saliva of infected cats. After bites, the virus persistently infect CD4 lymphocytes, where it is detectable as integrated proviral DNA 2-4 weeks post-infection (p.i.). During this Acute Phase (AP), non-specific symptoms (fever, neutropenia, anemia) can be detected. The viremia decrease to very low levels 2-4 months p.i., when circulating antibodies appear. This lead to the Asymptomatic Carrier (AC) state, lasting for several years, during which a Persistent Generalised Lymphadenopathy (PGL) might appear. In terminal stages, the lymphocyte infection lead to a progressive immunodepression (AIDS Related Complex or ARC) with the appearance of opportunistic infection such as skin lesions, stomatitis-gingivitis or of haematological disorders and to a final collapse of immune system (Feline AIDS or FAIDS). From the above reported brief description of the course of the disease arise that symptoms are non-specific and the diagnosis should be based on the detection of circulating antibodies or of proviral DNA. Antibodies can be detected during AC, PGL and, occasionally, ARC phases. Several in-house ELISA or immunochromatografic kits can be used to detect antibodies: doubt results should be verified by IFA or Western Blotting (gold standard). Proviral DNA can be detected by PCR since 2 weeks p.i. until the death. Real Time or TaqMan PCR might allow us to quantify the viral burden in order to monitor the progression of the disease. Similarly, the disease can be staged by measuring with a flow cytometer the CD4/CD8 lymphocyte ratio: CD4 lymphocytes, in fact, progressively decrease during the diseases, while CD8 lymphocytes decreases only in terminal stages.
2003
Settore VET/03 - Patologia Generale e Anatomia Patologica Veterinaria
European Society of Veterinary Clinical PAthology
State of the art: FIP and FIV diagnostics / S. Paltrinieri. ((Intervento presentato al 2. convegno 2nd CE day of the ESVCP-ECVCP tenutosi a Uppsala nel 2003.
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