Pneumonia in Cats associated with Neisseria sp.


 
 Neisseria sp. is a Gram-negative diplococcus bacterium usually present on the mucosal surfaces of animals without causing an obvious pathology. The objective of this study was to report the isolation of Neisseria sp. from severe cases of pyogranulomatous pneumonia with the formation of a Splendore-Hoeppli structure in two cats treated at a veterinary hospital. This paper suggests that the Neisseria genus members may be involved in lower respiratory tract infections in cats, with the molecular diagnosis being a necessary method for the correct identification of this bacteria in animals.


INTRODUCTION
The Neisseria genus is comprised of 32 species of gram-negative diplococci bacteria that are usually present on the mucous surfaces of humans and animals [1]. Neisseria gonorrhoeae, which causes gonorrhea [2], and N. meningitidis, which causes sepsis and meningitis [3], are the most prominent pathogenic species in humans.
In addition to these pathogens, it is recognized that this genus includes a wide variety of species such as Neisseria weaver, Neisseria dumasiana, Neisseria zoodegmatis, Neisseria shayeganii and Neisseria canis [4]. Some of them can induce disease [5], but most are identifi ed as commensals in the oral cavity and respiratory tract of humans and animals [4,6].
Few studies have been dedicated to the detection and characterization of Neisseria sp. in animals. More recent studies have reported the isolation of this bacteria in a case of deep dermatitis in a dog and of a nasofacial infection in a cat, and in the brains of geese and porpoises [7][8][9][10].
The objective of this study was to report the isolation of Neisseria sp. in severe cases of pneumonia in two cats treated at Hospital Veterinário, Brazil.

CASE PRESENTATION
Case 1. A cat, male, mixed breed, one-year-old, with a complaint of anorexia and dyspnea for approximately three days, was presented to a veterinary hospital. Chest radiography showed radiopacity with an interstitial and alveolar appearance in the lungs. The patient died on the same day.
Lung fragments were collected and sent to the microbiology laboratory where they were sown on 8% sheep blood agar (Sigma -Aldrich, Darmstadt, Germany), MacConkey agar (Neogen Corporation, São Paulo, Brazil), and Sabouraud Dextrose agar with and without chloramphenicol in aerobiosis, incubated at 37 °C, 30 °C, and 25 °C for up to 14 days for microbial and fungal isolation. Colonies were identifi ed according to a procedure described by Quinn et al. [11] by Gram stain, catalase, oxidase tests, and biochemical methods (Triple Sugar Iron-TSI, Indole Sulfi de Motility-YES, OF-GOF Test, Citrate) (Sigma-Aldrich, Darmstadt, Alemanha).
After isolation, each sample colony was inoculated into brain and heart infusion broth (Himedia Labs. Mumbai, India), incubated at 37 °C overnight, and geno mic DNA was extracted using the phenol / chloroform method [12].
Subsequently, the product obtained by PCR ( Figure 1) was purifi ed and sequenced using the ABI 3500 Genetic Analyzer (Applied Biosystems Foster, CA, USA). The sequence was analyzed by DECIPHER and compared to the GenBank database using BLAST (www.ncbi.nlm.nih.gov/BLAST) with access number MW354907. The sequence was similar to that of Neisseria sp. 97.35% (GenBank KC675471.1) and N. subfl ava 96.34% (GenBank LT689485.1). Phylogenetic analysis was performed on the Phylogeny.fr platform using the MUSCLE program (v3.8.31). After alignment, the ambiguous regions were removed with Gblocks (v0.91b) and the phylogenetic tree for the isolate was inferred by the maximum likelihood method (Figure 2).

Case 2.
A cat, female, mixed breed, fi ve years old, had a complaint of anorexia, dyspnea, and diarrhea for two days. On physical evaluation, mild jaundice, pulmonary crepitation, and moderate dehydration were observed, and replacement fl uid therapy  The patient died on the same day, and a diffuse dark red lung was observed at necropsy, with raised irregular and multifocal areas in the lung parenchyma ( Figure  3). Microscopically, a lung with marked changes in the parenchyma was observed, characterized by the respiratory tissue's replacement by a marked infl ammatory pyogranulomatous reaction. These were characterized by an aggregate of bacteria surrounded by degenerate polymorphonuclear cells with the Splendore-Hoeppli structure formation (Figure 3). Macrophages with a distended cytoplasm containing bacteria, similar to those that are aggregated outside the cell structure were observed. In almost all respiratory structures, the alveoli were distended, containing hyaline material, mononuclear cells, light infi ltrates of neutrophils, and bacterial colonies.
A macroscopic diffuse lobular pattern and a microscopic mononuclear, periportal and chronic infi ltrate with moderate fi brosis, along with proliferation of bile ducts,  were observed in the liver. In the bile ducts, morphological structures compatible with Platynossomum sp. and microabscesses were occasionally observed. There was microvascular hepatocellular degeneration, especially in the centrilobular region, and random individual coagulative necrosis of hepatocytes.
Microbiological analysis and molecular characterization of the isolate were similar to those described in the fi rst case (GenBank access number MW349688). The product generated by PCR showed a similarity of 99.22% with N. animaloris (MH166779.1) and 98.56% with N. canis (AY426974.1). Phylogenetic analysis is shown in Figure 3.

DISCUSSION
Species of the Neisseriaceae family remain part of the commensal microbiota of humans' and animals' upper respiratory tracts, usually without causing evident pathology [1,15]. In this study, we reported the isolation of Neisseria sp. in two cats with severe pyogranulomatous pneumonia.
In general, the diagnosis of bacterial pneumonia in felines occurs less frequently than in dogs [16]. The main microorganisms related to pathologies of the lower respiratory tract of cats include Pasteurella spp., Escherichia coli, Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Bordetella bronchiseptica, and Mycoplasma spp. [17].
Chest radiographs are commonly the fi rst test ordered when pneumonia is suspected [18]. In one of the animals in this study, the alveolar pattern was observed. In the other, the mixed pattern (interstitial and alveolar) suggested a picture of bacterial pneumonia as described by Levy et al. [19].
Initially, fungal pneumonia was considered in both cases as these patterns can also be seen in such conditions such as Pneumocystis canis and Penicillium brevicompactum infection [20,21]. Chest radiography is an auxiliary diagnostic tool; however, different diseases may reveal similar radiographic patterns [22].
The macroscopic and histological changes observed in this study were compatible with those described by Foster et al. [10], who investigated the infection by N. animaloris in porpoises (Phocoena phocoena). In both the cats, this multiple foci pattern indicated a hematogenous spread as described in porpoises after bites by gray seals (Halichoerus grypus).
Corroborating with other authors [7,9], confi rmation of the diagnosis was carried out through the isolation and sequencing of the 16S rRNA gene. According to Cobiella et al. [9], infection by Neisseria sp. should be considered whenever there is an isolation of gram-negative, catalase-positive bacteria that do not belong to the Enterobacteriaceae family, with a molecular diagnosis being a necessary method in the identifi cation and correct diagnosis of bacteria in humans and animals.
This study suggests that Neisseria genus members may be involved in lower respiratory tract infections (pyogranulomatous pneumonia) in cats.