A study of the relationship between feline infectious peritonitis and retroviruses in cats

02.2019, Translation 7.5.2021
Aydin Hakan, Yildirim Serkan
Original complete article: Investigation of the relationship between feline infectious peritonitis and retroviruses in cats

Abstract

Feline infectious peritonitis (FIP) infection is a highly pathogenic and fatal systemic inflammatory disease of cats. Although FIP infection is associated with the immune system, there are several predisposing factors for the disease. FIP has a high degree of interaction with other infectious agents. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections play a predisposing role by reducing immunity to FIP. This study was performed with Turkish Van cats, a cat breed specific to the Turkish Lake Van region, between 2014 and 2015. Ascites and signs of organ damage in 15 Van cats were identified by pathological analysis at Van Yüzüncü Yıl University. Polymerase chain reaction (PCR) was used to examine the presence of FIV and FeLV in the homogenate of brain, lung, liver, kidney, spleen and intestinal samples with lesions. FIV was identified in 1 (6.7%) of 15 cats and all cat samples were positive for FeLV (100%). Phylogenetic analysis of the positive samples revealed that it was endogenous FeLV. In the FIV phylogenetic analysis, our study strain was found to belong to the FIV subgroup B. The fact that FIP-positive cats were also positive for FeLV has drawn attention to the pathogenesis of endogenous-FeLV in malignancies, which has not yet been understood. In particular, it was concluded that the prevalence of FIP infection can be reduced by paying attention to the vaccination of young cats with FIV and FeLV in environments with a high cat population.

Introduction

Feline infectious peritonitis (FIP) is a fatal disease caused by the feline infectious peritonitis virus (FIPV), which is the result of a coronavirus mutation (FCoV). While FCoV has an enteric form, FIPV is a pathogenic type with a systemic infection. The dramatic increase in the cat population after World War II and the subsequent placement of cats in shelters paved the way for the most important scenario of the FIP. FCoV is thought to spread in the cat population due to their mass concentration, leading to mutant FIPV [1, 2]. Although the disease is not common in older cats, FIP infection is one of the leading causes of death in young cats aged 3 to 24 months. There are two types of FIP: effusive (wet) and granulomatous (dry). While the wet form is the most common and takes the form of inflammatory exudate in body cavities, the granulomatous form attacks the organs [1, 3]. The hypothesis of FIP is based on the fact that viral replication in cats with FCoV occurs in enterocytes. However, another variant of FIPV that can replicate in macrophages due to a mutation in the FCoV genome has also been published. [4]. FIP is a slow, progressive disease and can last for weeks or months. However, retroviral opportunistic infections such as feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), which cause immunosuppression, lead to the inevitable death by reduced immunity to FIP and accelerated disease. Although cats overcome FIP infection, secondary FeLV and FIV infection reactivates FIPV and dies in a short time. [4-6].

The Retroviridae family, which includes immune-suppressing viruses such as FIV and FeLV, has a linear, single-stranded, non-segmented, genome with a positive RNA polarity of 8-13 kb. Retroviruses that are morphologically enveloped and have icosahedral symmetry contain gag, pol and envelope gene regions [7, 8].

The immune system plays an important role in the clinical manifestations of FIP (wet / granulomatous). In granulomatous cases (dry FIP), T-cell mediated cellular immunity and humoral immunity are key. In FeLV infections in particular, FeLV infection affects the clinic of FIP infection due to disruption of this immune system mechanism. [9]. In addition, cats with FIP infection in the form of a latent or limited infection may experience re-infection due to FeLV. This will again result in death due to the FIP [5]. FIV infection is one of the most important co-infection factors in FIP outbreaks in cats with FCoV. It is reported that FCoV can achieve a very high rate of replication in immunosuppressed cats due to FIV infection, and that this may be a predisposing factor for FCoV to FIPV mutation. In cats infected with FIV and FCoV, FIV causes a high degree of FCoV replication and may lead to an outbreak of FIP in a short time. [1]. These retroviruses, which disrupt the immune system, are the most important clinical indicators of FCoV mutation. [1]. In the case of FIP-induced immune suppression, the transmission of opportunistic pathogens such as FIV / FeLV may occur. [10, 11]. Although there are currently commercial vaccines against FIV and FeLV, there is no protective vaccination or effective treatment against FIP in Turkey. [12].

This study was conducted to determine the interactions between FIPV infection and retrovirus co-infection (FIV / FeLV) in Van cats, a cat breed originating in Turkey.

Materials and methods

The population for the study consisted of a total of 15 cats with suspected FIP, which were transported to Van Yüzüncü Yıl University between 2014 and 2015 for pathological analysis of the death of the Turkish Van cat breed. Refrigerated tissue samples from the brain, lungs, liver, kidneys, spleen and intestines were sent refrigerated to a virology laboratory. For histopathological examinations, tissue samples from all tissues were taken into 10% formalin solution. After 48 hours of fixation in formalin solution, the samples were washed with running tap water for 10 hours. After passing through alcohol (70 °, 80 °, 90 °, 96 ° and 100 °) and xylene, they were immersed in paraffin blocks. Four thick sections were taken from each block to prepare slides on a slide. For histopathological examination, they were stained with hematoxylin-eosin (HE) and examined by light microscopy.

Immunohistochemical examination

All sections taken into adhesion (poly-L-lysine) immunoperoxidase slides were passed through xylene and alcohol series for deparaffinization and dehydration. They were then washed for 5 minutes in distilled water, 5 minutes with phosphate buffer (PBS, pH 7.2) and the endogenous peroxidase was inactivated by standing in 3% H202 for 3 minutes. After washing for 5-10 minutes in PBS, they were allowed to incubate for 5 minutes with a Protein Block that is compatible with all primary and secondary antibodies to prevent non-specific staining of the base. At the end of the incubation, the excess block solution remaining on the tissue sections was removed and drops of primary antibodies (VGEF, caspase 3, caspase 8, caspase 9, PCNA, neuronectin, TRPM 1, TRPM 2, α sinokline, apelin) and PBS in the control groups were used without washing. In accordance with the primary antibody, they were allowed to stand overnight at + 4 ° C. After washing twice for 5 min. with PBS, were incubated for 10-30 minutes. at room temperature with biotinylated secondary antibody. Sections washed again with PBS were allowed to stand in streptavidin-peroxidase for 10-30 minutes, then washed in the same manner with PBS. After washing, 3-3 'diaminobenzidine (DAB) chromogen was added to the sections and the mixture was allowed to stand for 5-10 minutes. according to chromogen intake. After immersion in Mayer's hematoxylin for 1-2 minutes for staining, they were rinsed with running water. They were then passed through a series of alcohol and xylene, covered with lamellae and examined by light microscopy (Leica DM 1000). Sections were rated negative (-), mild (+), moderate (++), and severe (+++) positive according to their immune positivity.

Investigation of retroviral infections in FIP positive cats

For FIV and FeLV DNA testing of FIP-positive cats, liver, spleen, kidney and brain tissue samples with lesions were taken according to pathological analysis. These tissue samples were combined to prepare a homogenate. Nucleic acid isolation was performed using a supernatant from this tissue homogenate using a commercial kit (GF-1 Nucleic Acid Extraction Kits, Vivantis, Malaysia). The resulting nucleic acid suspension was used as a template for PCR for FIV and FeLV analysis. For FIV DNA screening, nested-PCR was performed using primer pairs specific for the V3-V6 region of the env gene (Table 1). [13]. For FeLV detection, PCR was performed using primer pairs specific for the FeLV env gene region [14]. The primer sequences and product sizes are listed in Table 1.

Example5′-3 ′ sequencePolaritySpecificitySize (bp)
Env FwTAY TGG GCC TGT AAC ACY G+FeLV508
Env RvCGC TGT TTT AGT CTT TCT CTT A-FeLV
VE1SGAG TAG ATA CWT GGT TRC AAG+FIV1211
VE1RCAT CCT AAT TCT TGC ATA GC-FIV
nVE2SCAA AAT GTG GAT GGT GGA AY+FIV859
nVE2RACC ATT CCW ATA GCA GTR GC-FIV
Table 1: Details of oligonucleotides used in the study

Sequential and phylogenetic analysis

After gel electrophoresis, FIV / FeLV positive PCR products were subjected to sequence analysis. The raw data obtained after sequencing were confirmed by GenBank (NCBI National Center for Biotechnology Information / BLAST). Sequences of reference strains and studied samples were arranged by BioEdit program (version 7.0.5) [15]. Phylogenetic analysis was performed using the MEGA program (version 6.0) by the neighborhood method [16].

The results

Pathological findings

At necropsy, gray-white granulomatous lesions in the spleen, liver, kidneys, intestines, lungs and cerebral organs and serous atrophy were observed macroscopically in adipose tissue (Figure 1). In the effusion form, the mucous membranes were found to be strongly icteric and there was a discharge of 1-1.5 l in the abdominal cavity (Figure 2).

Figure 1: Non-fusion form, pyogranulomas clearly visible in seroses of liver, spleen and intestine
Figure 2: Efusion form, approximately 1.5 l of effusion fluid in the abdominal cavity

Microscopic examination revealed, especially in the non-fusible form, pyogranulomatous lesions, necrotic in the middle, spleen, liver, kidneys, intestines, lungs and brain tissues, surrounded by neutrophils, macrophages, lymphocytes and plasma cells (Figure 3). Vasculitis was found in the vessels in these organs (Figure 4). While these granules were generally observed near the serosa, some of them progressed to parenchymal tissue, especially in the kidneys.

Figure 3: Liver, pyogranuloma and mononuclear cell infiltrations consisting of neurophilic leukocytes and mostly with necrotic mass in the middle; H&E, Molding: 50 µm
Figure 4: Renal tissue, FIP positive in tubular epithelium and interstitial intermittent macrophages, IHC-P, Rail: 100 µm

Virological findings

FIV / FeLV DNA was examined by molecular methods in 15 Van cats with FIP diagnosed by pathological analysis. As a result of PCR analysis, FeLV DNA was detected in all 15 cats (100%). In the phylogenetic analysis of FeLV strains in our study and GenBank reference strains, FeLV strains formed two phylogenetic groups: exogenous and endogenous. Sheep and horse retroviruses have been used as a non-member group. In phylogeny, it was observed that all our study strains coalesced together with endogenous FeLV (enFeLV) (Figure 5).

Figure 5: Phylogenetic tree based on the 508 bp nucleotide sequence of 15 FeLV strains generated by Neighbor-Joining using MEGA 6.0 software. Equine and ovine retroviruses were used as a non-member group. Round symbols (•) indicate Turkish tribes

PCR analysis identified FIV in 1 (6.7%) from 15 samples. Subsequently, a sequential reaction and phylogenetic analysis of the FIV strain was performed. Phylogenetic analysis of the FIV strain obtained from our study and reference strains from GenBank showed that the FIV strains were divided into 6 different subtypes from A to F. Our FIV strain was classified as FIV subtype B along with Turkish and Japanese strains (Figure 6).

Figure 6: Phylogenetic tree based on the 859 bp nucleotide sequence of FIV strains generated by Neighbor-Joining using MEGA 6.0 software. The square symbol indicates a Turkish tribe

Discussion

FIP is a systemic infection associated with the immune system caused by coronaviruses. This insidious and deadly infection significantly threatens domestic and street cats that share the same environment with humans, and even breeds of protected cats such as Van cats. Eliminating predisposing factors is the first combat strategy against FIP infection, for which there is not yet an effective method of prevention and treatment in Turkey. Of these factors, cleaning, hygiene and negative interaction in ordinary living spaces are important. Immunosuppressive diseases, such as FIV and FeLV, are the most important predisposing factors for the development of FIPV, a mutant form of coronavirus.

In our study, pathological analysis revealed FIP infection in cats of Van, a breed native to Turkey. Subsequently, the presence of FIV and FeLV was examined in tissue samples obtained from these cats.

FeLV and FIV are pathogenic infectious agents that cause immunosuppression mortality in domestic cats. While FIV is similar to human HIV infection, FeLV causes immunosuppressive, neoplastic, and hematopoietic disorders. [17]. Retroviral infections of cats are the most important trigger for FIP infection in cat homes and shelters. Successful results against retroviral infections have been achieved thanks to the aggressive policies of many Western countries, such as the eradication of infections through testing and vaccination. [18]. Although vaccines against FIV / FeLV infections exist in Turkey, an adequate and controlled vaccination program has not yet been established.

In our study, FeLV was detected in all cats with FIP. FeLV is divided into 6 groups: A, B, C, D, E and T. These types, called exogenous FeLV, are responsible for leukemia and tumor formation in cats. Endogenous FeLV (enFeLV) are FeLV sequences transfected into the genome of domestic cats. enFeLV proviruses are found in various organs and tissues and perform translation and transcription. However, due to the lack of essential parts of the viral genome, it cannot become an infectious viral particle. However, infection of a cat with exogenous FeLV (field strain) leads to recombination between the two (endogenous / exogenous) and transforms enFeLV into an infectious virus particle. However, it is still unclear whether enFeLV may be responsible for malignant formations in cats with lymphoma where exogenous strains are not detected. [19].

In our study, FeLV was detected in all tissue suspensions with pathological lesions collected from Van cats. Phylogenetic analysis showed that they were all enFeLV. Although this suggests that enFeLV may mutate and cause malignant formations, there is no information in the literature to support this hypothesis. There are several studies on coronavirus infection of cats in Turkey. In a study by Pratelli et al., FCoV seropositivity was determined in 62% cats living in environments such as shelters. [20]. In a study by Sahna et al. by molecular methods, the rate was identified by FCoV 54% [21]. A study in Turkey identified a rare ocular form of FIP infection in cats [22]. In a retrospective study of FCoV, FIV and FeLV in 169 sick cats in Turkey, seroprevalence ratios were determined to be 37%, 11% and 1% [23]. Similarly, in a study investigating FCoV, FIV and FeLV in chronic gastrointestinal, urinary, symptomatic and healthy cats in Turkey, viral substances were detected in 45.5%, 9.5% and 20.5%, respectively [24]. In our study, one cat was found to be co-infected with FIV and FeLV. In the phylogenetic analysis of the FIV strain identified in our study, it was found to be a subtype group B. In a previous study performed in cats in Turkey, subtype B was identified in parallel with our results. [25]. In a study of FIV and FeLV infections in cats, the incidence rates of FIV and FeLV were found to be 22.3% and 5.8%, respectively. [26]. Because our study was limited to a specific group, a relatively low FIV / FELV rate was achieved.

Conclusions

Eliminating predisposing factors is the first combat strategy against FIP infection, for which a method of prevention and treatment is not yet effective in Turkey. Although there are vaccines against FIV / FeLV infections that are predisposing factors for FIP, an adequate and controlled vaccination program has not yet been established. Due to the increased incidence of FCoV and predisposing factors, vaccination and awareness of cat owners is important in this regard in order to minimize the spread of FIPV. There is a need to establish a national eradication and control policy for agents such as FIPV and FeLV, which prepare the ground for FIPV infection in shelter cats and even in protected cat breeds.

Compliance with ethical standards

Acknowledgement

SY: For a time he worked at Van Yüzüncü Yıl University. At that time, cat samples were obtained. SY: provided cat samples and performed pathological analyzes, HA: performed virological analysis. SY and HA read the whole article and agreed.

Disclosure of conflicts of interest

The authors declare that they are not aware of the conflict of interest.

Declaration of ethical approval

This study was approved by the Ethics Committee of the Veterinary Faculty Unit of Van Yüzüncü Yıl University (Protocol No. 2015/03).

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