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Regulatory Mechanisms in JtSiosystems
¿m' v* ' Jfflfc Regulatory Mechanisms ISSN 2519-8521 (Print) ISSN 2520-2588 (Online)
Regul. Mech. Biosyst.,
in Biosystems 2019, 10(4), 507-512
doi: 10.15421/021974
Pathogenetic role of Staphylococcus aureus in purulent keratoconjunctivitis in cats
S. N. Maslikov, D. D. Bely, V. V. Samoiliuk, V. V. Vakulik, T. L. Spitsyna
Dnipro State Agrarian and Economic University, Dnipro, Ukraine
Article info
Received 12.10.2019 Received in revised form
08.11.2019 Accepted 09.11.2019
Dnipro State Agrarian and Economic University, S. Efremov st., 25, Dnipro, 49600, Ukraine. Tel.: +38-097-252-62-73. E-mail:
maslikovs.62@ukr.net
Maslikov, S. N., Bely, D. D., Samoiliuk, V. V., Vakulik, V. V., & Spitsyna, T. L. (2019). Pathogenetic role of Staphylococcus aureus in purulent keratoconjunctivitis in cats. Regulatory Mechanisms in Biosystems, 10(4), 507-512. doi:10.15421/021974
The research was carried out in the Department of Surgery and Obstetrics of Agricultural Animals of Dnipro State Agrarian and Economic University on clinically healthy outbred cats of different ages with purulent keratoconjunctivitis. Hematological, biochemical and immunological parameters were determined in the animals, and microbiological and virological research was conducted on them. According to the data obtained, more than half of cases of ophthalmopathology in cats were conjunctivitis and keratoconjunc-tivitis, and they were more often registered in the cold season. The main causes of eye diseases in the cats were mechanical injuries, coccal and chlamydial infection, allergy and development of disease against the background of primary lesions of the ears and paranasal sinuses. Among the detected microorganisms, the vast majority (81.9%) were staphylococci, including S. albus, S. aureus and S. epidermidis. All types of microorganisms except O-forms of Bacillus sp. exhibited high and medium sensitivity to antibiotics. Immunoblotting revealed polypeptides that responded to specific S. aureus antigens in samples of conjunctiva, cornea, intraocular fluid, and blood of cats suffering from purulent (staphylococcal) keratoconjunctivitis. The highest antigen concentration was detected in the cornea and conjunctiva. In the absence of expressive shifts of the investigated hematological and biochemical parameters, the dynamics of immunological markers were shown by a significant increase in the content of immunoglobulins and circulating immune complexes, as well as by a distinct activation of the complement system by the classical route. The results showed a clear gradual activation of phagocytosis, namely: the number of phagocytic neutrophils increased, reaching its maximum value by the seventh day of development of purulent keratoconjunctivitis. The phagocytic index in the first three days of observation tended to decrease, and by the seventh day it had already significantly exceeded the initial value. Despite the ambiguous dynamics of the phagocytic index, in the course of the development of the inflammatory process in the blood of sick cats, we observed a clear increase in the index of complete phagocytosis. Thus, the development of purulent keratoconjunctivitis in cats occurs against the background of clear cellular and humoral responses to the infectious agent.
Keywords: cats; ophthalmopathology; diagnostics; microorganisms; immunoblotting.
Comparison of genetic markers indicates the widespread distribution of identical S. aureus strains among animals (36.3%) and veterinary staff (38.9%) with the presence of PVL-positive clone home companions, which are a possible source of transmission to humans (Drougka et al., 2016). S. aureus is capable of infecting the lacrimal duct, eyelids, conjunctiva, cornea, anterior and posterior chambers of the eye, as well as the vitreous, causing loss of vision or even blindness (O'Callaghan, 2018). Importantly, there is no difference in the species composition of the eye microflora between clinically healthy animals and patients with conjunctivitis, although in the latter case S. epidermidis and S. aureus were most commonly isolated (Kielbowicz et al., 2015). A considerable number of reports have been devoted to identifying bacterial associations of the conjunctival sac. In particular, Espinolaz & Lilenbaum (1996) emphasize the importance of the species S. felis, although they indicate a significant prevalence of S. epidermidis (45.7%), S. simulans (23.9%), S. auricularis (17%) and S. saprophyticus (6.5%) ). Aftab et al. (2019) note also the high presence in the conjunctival sac of S. pyogenes (18.9%) and Escherichia coli (11.5%). A significant increase in the prevalence among cats resistant to antibacterial agents of pathogenic Staphylococcus species, in particular S. aureus, has been demonstrated (Lane et al., 2018). Insufficient study of pathogenesis of conjunctivitis in cats is confirmed by the results of modern observations, in which, based on the analysis of the 16S rRNA gene sequence, live taxa previously undescribed in veterinary medicine were identified ; S. caprae, S. succinus, Propionibacterium acnes, P. faecalis and Bacillus subtilis (Ploneczka-Janeczko et al., 2017). Unlike in Ukraine, this issue is receiving considerable attention in foreign countries. Based on
Introduction
Conjunctivitis in cats is characterized by a multitude of possible etiological factors, including the herpes virus (FHV), Chlamydophila felis, mycoplasma and aerobic bacteria. Among the latter, the most common species are Staphylococcus, Streptococcus and Micrococcus (Hartmann et al., 2010). In particular, according to Buttner et al. (2019), microorganisms that are the cause of conjunctivitis were isolated in 40.8% of clinically healthy cats. Gram-positive bacteria accounted for 71%, gram-negative bacteria - 26%, fungi - 3%, with the highest prevalence being for S. felis (19.8%) andM. osloensis (5.8%). For many years, S. aureus was thought to occur only in humans. Recent studies indicate its prevalence in both domestic and farm animals, which can be a reservoir of infection against the background of proven pathways for transmission in humans (Biero-wiec et al., 2014; Kock et al., 2014). S. aureus in both human and veterinary medicine is recognized as a significant pathogenetic factor in the development of the disease. Given the isolation of S. aureus isolates at 17.5% and methicillin-resistant (MRSA) in 6.5% of clinically healthy animals, Bierowiec et al. (2016), the risk factors for the infection include the work of the owners in the field of health care, work in veterinary medicine and the fact that the cats were treated with antibiotics during the year. Numerous reports indicate that cats may play a role in the transmission of methicillin-resistant S. aureus (MRSA) to humans, but the lack of data on its prevalence in animals makes it impossible to study this process in detail (Bramble et al., 2011). In this case, pets and humans are susceptible to infection with both S. aureus and MRSA (Kottler et al., 2010).
the dangers for humans of S. aureus isolated from conjunctivitis in cats, and given its lack of sensitivity to many antibacterial agents, the World Association of Veterinary Dermatologists (WAVD) offers recommendations for diagnosis, therapy, and hygiene and disinfection for staphylococcal infection (Morris et al., 2017). Thus, most authors are limited to isolating isolates without determining its effect on the body, in particular immune status. It should be noted that studies on the prevalence of conjunctivitis caused by S. aureus in cats and their role in human morbidity due to transmission from pets have not been conducted. In addition, the pathogenetic aspects remain insufficiently studied, which determines the relevance of the study of this pathology. Based on the above, the aim of the study was to study the prevalence of conjunctivitis in cats and the role of S. aureus in the pathogenesis of this zoonotic disease.
Material and methods
The research was carried out at the Department of Surgery and Obstetrics of Agricultural Animals of Dnipro State Agrarian and Economic University, two state (Samara, Shevchenkivsky and Soborny districts) and two private (FOP Alimov and PE "Vetol") veterinary medicine clinics in Dnipro between 2015 and 2018, in accordance with the requirements of the "European Convention on the Protection of Vertebrate Animals" (Strasbourg, 1986) and the Law of Ukraine on the Protection of Animals Against Cruelty (2006). The protocol of the research programme was approved by the conclusion of the Commission on Bioethics of the Dnipro State Agrarian and Economic University. Monitoring studies were performed by analyzing the records of the registration of sick animals in the outpatient journals of these institutions. Clinical studies were made in the Department of Surgery and Obstetrics for Animals; laboratory studies were performed atf the Research Center for Safety and Environmental Control of Resources of the Agrarian-Industrial Complex of the Dnipro State Agrarian and Economic University and the Dnipro Regional Veterinary Laboratory on 16 clinically healthy animals of 1 day, 3 months and 3 years old; 5 clinically healthy and 10 outbred cats with purulent keratoconjunctivitis aged 12-14 months, weighing 3.5-4.0 kg.
Animals were kept in individual cages measuring 0.9 m2 throughout the study period. Before the experiment, the animals were dewor-med (cat Drontal), their clinical status, hematological and biochemical serum levels were determined, washings were taken from the conjunc-tival bladder for seeding on nutrient media (meat-peptone broth, sucrose broth-peptone agar, sucrose agar, staphylococcal agar No 110, Saburo agar with chloramphenicol and cycloheximide). Species belonging to staphylococci were determined by lecithinase plasma-coagulating, hemolytic activity and decay of mannitol under anaerobic conditions. Antibiotic sensitivity was determined using paper discs. In the blood of the animals we determined indicators of morphological status: leukocytes - melangery method in the Goryaev counting chamber; the leukocyte formula was deduced by counting 200 leukocytes in smears stained with the Romanovsky-Gimza. The phagocytic activity of neutrophils (PAN) of peripheral blood was studied by a test method (from S. epi-dermidis strain 9198), followed by counting the phagocytic number in stained smears, index of completed phagocytosis. The nitrosine tetrazo-lium spontaneous test was evaluated by the reduction of nitrosine tetra-zolium. The percentage of T-lymphocytes, their subpopulations and B-lymphocytes was determined by the reaction of rosette formation with erythrocytes, which adsorbed monoclonal antibodies against receptors CD3 (T-lymphocytes), CD4 (T-helper), CD8 (T-suppressors), CD16 (natural closets), CD 19 (B-lymphocytes) (Lora et al., 2000). The liver biosynthetic function was evaluated by the level of total protein (refracto-metrically) and protein fractions (nephelometric method). The content of class A, E, G, and M immunoglobulins was determined by the enzyme immunoassay and the circulating immune complexes by the polyethylene glycol precipitation method (Grinevich et al., 1981). The activity of total complement was evaluated by its hemolytic activity (50% hemolysis) in a unified method with ram erythrocytes in the presence of rabbit serum. The activity of the complement component C3 was determined by the enzyme-linked immunosorbent assay, and the complement component C4 by the immunoturbidimetric method on a Cobas 6000 analyzer.
The presence and distribution of polypeptides that reacted with specific S. aureus antigens were determined in conjunctiva and corneal ho-mogenates, chamber fluid, and cat blood by electrophoresis and immu-noblotting. Electrophoresis of protein antigens was performed in poly-acrylamide gel by the method of Laemmli et al. (1970). Electrophoretic transfer of antigens from the gel to the nitrocellulose membrane was carried out by the method of Towbin et al. (1979). As a positive control, we used reference rabbit serum to S. aureus (strain 209).
The work used the program "LabWork 4.0" (uVp, 2001) to scan and compare the intensity of polypeptide zones on the nitrocellulose membrane after immunoblotting. The colour intensity of the zones of S. aureus polypeptides in the control group was taken as 1 (100%). Conditional units in which the S. aureus content was expressed were calculated by assigning the relative zone density (%) to the protein content in the sample (^g).
Cats with purulent keratoconjunctivitis were subjected to the same investigation procedures on the first, third and seventh days of the disease, but prior to the experiment, they were sampled (serum and scrapers) for analysis for herpes (FHV-1), adenovirosis, chlamydia, mycoplasmosis and toxoplasmosis by real-time polymerase chain reaction and enzyme immunoassay.
Statistical processing of the results was performed using Statistica 10 (StatSoft Inc., USA, 2011). A Bonferroni-corrected ANOVA was used to determine the difference between the samples.
Results
In the veterinary clinics involved in the research, an average of 1,045 animals are registered annually, among which 41.5% are cats suffering from surgical pathology, and the incidence is higher among males. In each of the veterinary hospitals, 7.7% of sick cats have eye diseases. Metis (73.2%) make up the vast majority of patients. At the same time, ophthalmic pathology is most commonly reported in cats of Persian, British breeds and sphinxes.
In the structure of ophthalmic pathology, conjunctivitis accounts for 6.7% of the number of patients with surgical diseases and 35.5% - of the total number of eye diseases (Fig. 1). In 81.0% of cases, conjunctivitis is diagnosed with bilateral lesions, and 39.2% of animals have puru-lent-catarrhal disease. In most cases, the cause of the lesion is coccal flora colonization (staphylococci), the remaining cases are allergic. Unilateral conjunctivitis usually occurs as a result of trauma, but in some animals the etiology of the disease is not clear.
Keratoconjunctivitis is one of the most commonly reported eye pathologies. 5.5% of surgically ill animals are diagnosed with this disease, but keratoconjunctivitis accounts for 29.2% among eye diseases.
3.3 0.9
3.3
29.2
□ conjunctivitis
□ keratitis
□ keratoconjunctivitis
□ panophthalmitis
□ iridocyclitis
□ retrobulbar
phlegmon
□ cataract
□ glaucoma
□ corneal
sequestration
□ blepharitis
Fig. 1. Nosological profile of ophthalmopathology in cats in Dnipro (2015-2018, %, n = 1323)
Keratoconjunctivitis occurs in the form of purulent catarrhal or serous catarrhal inflammation and in the absolute majority of cases (84.4%) is bilateral. The primary etiological factor of the disease is the primary colonization of the coccal flora or its development on the back-
ground of an allergic condition, in particular with paranal adenitis, although, in 15.6% of the animals the disease is traumatic in origin. It is also interesting that 6.7% of cases of keratoconjunctivitis have a chlamydial etiology - four cases of chlamydiosis of the eyes are registered in two private hospitals, according to which the proportion of chlamydial keratoconjunctivitis is 21%. Chlamydia is not recorded in other veterinary hospitals, possibly because of the lack of conditions for diagnosis.
Cat diseases of the eyes are mostly recorded in the cold season. By seasonality, the incidence is as follows: winter - 41.6%; spring -23%; summer - 9.5%; autumn - 25.9%. More detailed analysis revealed that males are more likely to fall ill in winter and females are more likely to fall ill in the autumn. Ophthalmic pathology is diagnosed in patients aged one month to eighteen years, in particular: up to 3 months -15.8%, 3-6 months - 9.0%, up to one year - 11.6%, three to five years - 16%, ten years - 11.9%, more than 10 years - 17.4%.
The conjunctival cavity of healthy cats is a harbour of a variety of microorganisms, including S. albus, S. aureus, Bacillus sp., that enter the conjunctival bladder from the external environment, however, humoral and cellular defense factors are able to control their pathogenic effects. It should be noted that superficial mechanical scarification of the connective eye tissue does not cause clear clinical signs of inflammation (tear, light phobia and blepharospasm were absent). Only scarification sites have superficial moderately hyperemic erosions that gradually decrease and completely epithelialize for 8-9 days, even without treatment.
The seeding of material from the conjunctiva of clinically healthy cats in nutrient media in all cases showed an increase in microflora, including: 1 case of Bacillus sp. (O-form), which appeared to be insensitive to antibiotics; 1 case of Bacillus sp. (R-form), which was sensitive to antibiotics with a growth inhibition zone, respectively: gentamicin -20 mm, levomycetin - 15 mm, tetracycline, norfloxacin and enroxil -12 mm; 4 cases of S. albus, which showed sensitivity to antibiotics with growth inhibition zone, respectively: ampiclox - 30 mm, ceftriofur -30 mm, clamoxyl - 29 mm, lincomycin - 24 mm, levomycetin -23 mm, floran - 22 mm; 6 cases ofS. epidermidis susceptible to antibiotics with growth inhibition zone, respectively: ampiclox and clamoxyl -30 mm, ceftriofur - 28 mm, floran - 24 mm, gentamycin - 20 mm, lincomycin - 17 mm; 4 cases of S. aureus, which are sensitive to antibiotics with growth inhibition zone, respectively: ampiclox - 31 mm, ceftriofur - 30 mm, clamoxyl - 30 mm, lincomycin - 28 mm, levomy-cetin - 25 mm, floran - 22 mm. Among the detected microorganisms, the vast majority were staphylococci, including S. albus and S. aureus accounted for 25%, and S. epidermidis - 37.5%
In the conjunctival smears of clinically healthy cats, a positive response to C. psittaci was detected in 3 animals by real-time polymerase chain reaction. In addition, one of the chlamydia-responsive animals showed an even more positive response to M. felis.
The purulent inflammation of the conjunctiva and cornea is manifested by rather pronounced local symptoms with varying degrees of visual impairment. However, according to current concepts of inflammation, this pathological process should be considered conditionally local. From this point of view, the data we have obtained regarding the distribution of pathogens in the body and their effect on the function of other organs and systems are of particular interest. Samples of the conjunctiva, cornea, intraocular fluid and blood of cats suffering from artificially induced purulent (staphylococcal) keratoconjunctivitis revealed polypeptides that respond to specific S. aureus antigens.
The test specimens exhibit characteristic differences in the content of S. aureus antigens, which is evidence of the differential distribution of the infectious agent, probably due to the complex of circumstances and conditions, including the characteristics of the investigated substrates and tropism of the pathogen (Fig. 2). The highest concentrations of S. aureus antigen were detected in the cornea (58.2 ^g) and conjunctiva (38.2 ^g). Intraocular fluid and blood receive respectively 14.4 and 7.2 ^g of the infectious agent.
The dynamics of immunological parameters (Table 1) show a significant (except IgG) increase in the content of immunoglobulins and CIC: IgA - by 102.0%, IgE - by 40.3%, IgM - by 54.2%; circulating immune complexes - by 64.2%, as well as a clear activation of the complement system. On the seventh day, similar changes in the level of
circulating immune complexes (by 74.5%) and immunoglobulins were observed, but the increase in IgG level (by 36.2%) became significant (P < 0.05). In cats, there is an unreliable increase in the activity of total complement, with the maximum indicator (115.5%) reached on the third day of observation. The dynamics of the C3-component are fluctuating and unreliable, namely: on the first day of research the level of C3 increased by 2.5%, on the third day it was 8.9% less than the initial one, and on the seventh day it again exceeded it by 3.8%. The indicators of C4 almost repeat the overall dynamics of C3, but on the third day there was a significant decrease (P < 0.05).
The number of phagocytic neutrophils gradually increased by 10.218.4% (Table 2), reaching its maximum value by the seventh day of development of purulent keratoconjunctivitis (133.6%). The phagocytic index in the first three days of observation tended to decrease (by 7.016.3%), and by the seventh day it already significantly exceeded the original value by 76.7% Despite the ambiguous dynamics of the phago-cytic index, an increase in the index of complete phagocytosis (up to 50.0% on the seventh day) was observed in the course of the development of the inflammatory process in the blood of sick cats.
80 T 75 -70 -65 -60 -- 58.2***°°° 55 -50
40 -35 -30 -25 20
15 -10 -5
0
38.2**°°°
14.4°
m
7.2
cornea conjunctiva chamber fluid blood
Fig. 2. Distribution of S. aureus in blots of the cornea, conjunctiva, internal fluid, and blood of Dnipro cats suffering from purulent keratoconjunctivitis (2015-2018, n = 10); ° - P < 0.05; ** - P < 0.01;
*** °°° - p < 0.001, about content: * - in the chamber fluid, ° - blood by comparison with the Bonferroni-corrected ANOVA
Table 1
Dynamics of indicators of humoral immunity
of cats suffering from purulent keratoconjunctivitis (x ± SD)
Animals
Indicators
clinically healthy, n = 5
patients with purulent keratoconjunctivitis, n = 10
1 day_3 day
7 day
Total protein, g/L 60.2 ± 1.10 62.0 ± 1.20 63.0 ± 1.10 65.0 ± 1.10
Albums, g/L 29.0 ± 0.97 29.0 ± 0.98 27.7 ± 1.00 29.0 ± 1.20
Globulins, g/L 30.2 ± 0.89 32.0 ± 0.87 36.0 ± 1.40 36.0 ± 1.50
IgA, gL 2.0 ± 0.19 2.3 ± 0.07 4.0 ± 0.52* 3.8 ± 0.33
IgE, IU/mL 2.9 ± 0.04 3.4 ± 0.11 4.1 ± 0.06 4.0 ± 0.10*
IgG, g/L 6.9 ± 0.19 7.1 ± 0.15 7.4 ± 0.14 9.4 ± 0.37*
IgM, gL 2.1 ± 0.28 2.5 ± 0.13 3.3 ± 0.11* 3.6 ± 0.11*
Circulating immune complexes, conventional units 28.0 ± 2.70 30.0 ± 1.79 46.0 ± 1.60* 49.0 ± 1.40*
Total complement activity, conventional units 57.0 ± 2.80 62.0 ± 2.40 66.0 ± 1.20 63.0 ± 1.30
C3 component of complement, g/L 0.80 ± 0.02 0.80 ± 0.02 0.70 ± 0.02 0.80 ± 0.02
C4 component of complement, g/L 0.30 ± 0.02 0.30 ± 0.01 0.28 ± 0.01* 0.30 ± 0.01
Note: * - P < 0.05 relatively clinically healthy animals. Discussion
Conjunctivitis, along with keratoconjunctivitis and corneal ulceis, is widespread not only in humans but also in animals (Spadea et al., 2018), as confirmed by our observations in Dnipro. Ophthalmic diseases are
accompanied by similar clinical features (Giudici & Pressanti, 2014), so determining the etiological factors that may be represented by zoonotic pathogens plays an important role in the complex diagnosis and development of treatment interventions (Gerding et al., 1990).
Table 2
Dynamics of indicators of cellular immunity
of cats suffering from purulent keratoconjunctivitis (x ± SD)
Animals
clinically patients with purulent healthy, keratoconjunctivitis, n = 10 n = 5_1 day_3 day_7 day
Leukocytes, G/L 7.9 ± 0.42 8.0 ± 0.27 8.5 ± 0.19* 8.4 ± 0.11
Eosinophils, % 3.4 ± 1.08 3.5 ± 0.67 3.7 ± 0.22 3.8 ± 0.26
Rod-core, % 3.2 ± 0.73 3.8 ± 0.26 3.6 ± 0.23 4.0 ± 0.22
Segmented nuclear, % 54.0 ± 1.80 53.0 ± 1.60 56.0 ± 1.30 54.0 ± 0.94
Lymphocytes, % 39.0 ± 2.30 39.0 ± 1.60 35.0 ± 1.08 36.0 ± 0.90
Monocytes, % 0.7 ± 0.32 1.1 ± 0.25 2.3 ± 0.16* 2.2 ± 0.14*
CD3, % 52.0 ± 2.80 48.0 ± 1.70 45.0 ± 1.20* 38.0 ± 1.90*
CD4, % 34.0 ± 1.90 29.0 ± 0.89 30.0 ± 0.89 29.0 ± 1.20
CD8, % 21.0 ± 2.3 25.0 ± 0.83 28.0 ± 1.03 30.0 ± 0.66
CD16, % 22.0 ± 2.20 29.0 ± 1.04 31.0 ± 0.94* 32.0 ± 1.50*
CD19, % 22.0 ± 0.86 23.3 ± 0.90 22.0 ± 1.09 20.5 ± 1.30
Spontaneous NBT, % 4.5 ± 0.15 4.6 ± 0.32 5.9 ± 0.44* 4.9 ± 0.16*
Phagocytic activity of neutrophils, % 34.0 ± 3.30 38.0 ± 1.30 40.5 ± 1.50* 46.0 ± 1.20*
Phagocytic number 4.3 ± 0.32 4.0 ± 0.31 3.6 ± 0.17 7.6 ± 0.32*
Index of complete phagocytosis 1.8 ± 0.21 2.2 ± 0.14 2.5 ± 0.18* 2.7 ± 0.16*
Note: see Table 1.
Statistical analysis showed a significant prevalence of conjunctivitis in domestic cats, which is consistent with reports from other researchers (Ploneczka-Janeczko et al., 2017), who also believe that in addition to infectious factors, this disease can be caused by trauma, hypersensitivity reactions, etc. The conjunctival sac has been found to be inhabited by more diverse associations of microflora than previously thought, the most common of which are cat herpes virus (FHV-1), C. felis and M. felis, causing serous mucous-purulent discharge from the eye. Hillstrom et al. (2012) argue that cytologic examination of conjunctivitis of cats allows one to determine the type of inflammation and to detect some microorganisms, in particular C. felis, but their results are contradictory.
The occurrence of hospital infections in veterinary clinics, in particular staphylococcal (Quitoco et al., 2013) and the widespread use of antibacterial therapy are among the main causes of the high likelihood of human infection, especially from the point of view of multiple pharmacological resistance of microorganisms (Palchykov et al., 2019; Za-zharskyi et al., 2019). It is therefore advisable to conduct training programmes for monitoring not only productive but also small animals (Wieler et al., 2011). Because MRSA lines isolated from infected pets often reflect epidemic human strains circulating in the same region a successful disease control strategy requires coordinated efforts by human and veterinary medicine, in line with the "Single Health" concept (Vincze et al., 2014).
In this case, Worthing et al. (2018) indicate that the transmission of S. aureus and S. pseudintermedius by veterinary staff to animals and vice versa is restricted in non-outbreak conditions.
Human medicine has proven that S. aureus is a major causative agent of eye diseases, capable of infecting the lacrimal duct, eyelids, conjunctiva, cornea, anterior, posterior, and vitreous chambers of the eye. In addition to causing skin and soft tissue infections, osteomyelitis, endocarditis, sepsis and pneumonia, S. aureus is one of the most common causes of eye infections, including blepharitis, dacryocystitis, conjunctivitis, keratitis and endophthalmitis (O'Callaghan, 2018). S. aureus is thought to accelerate the development of experimental allergic conjunctivitis (Chung et al., 2009) and to activate conjunctival inflammation (McGilligan et al., 2013). The virulence of different strains of S. aureus for the conjunctiva varies (McCormick et al., 2011). But as far as cats are concerned, these issues are still poorly understood today.
Infection of the external structures of the eye is one of the most common types of diseases (Armstrong, 2007). Bacterial conjunctivitis is increasingly becoming an independent disease that can be caused by "normal" microflora, in particular S. aureus, or by its pathogenic strains (Diamant & Hwang, 1999), which is consistent with the results of our studies. The studies conducted add to and confirm the message (Weese et al., 2015) that associations of microorganisms that populate the conjunctiva play an important role in the protection against pathogenic infections and at the same time are a source of potential pathogens. The microbial flora of the conjunctiva interacts closely with the immune system, and changes in an animal's immune status can lead to some changes in this association of microorganisms. Pathogens are staphylo-cocci, which are widespread on mucosal surfaces and can be associated with a wide range of infections, including conjunctivitis. Therefore, we can assume that this microflora is not normal for the conjunctiva. As our research has shown, primary bacterial eye infections associated with corneal and conjunctival lesions are quite common.
The high incidence of staphylococci and their role in the pathogen-nesis of eye diseases in cats confirms the results presented by other researchers (Adler et al., 2007; Lin & Petersen-Jones, 2008), which is explained by the presence in the conjunctival sac of healthy animals of S. aureus, S. saprophyticus, Bacillus sp., E. coli and Enterobacter sp. (Cardoso et al., 2012). The sensitivity to antibacterial agents is consistent with the data of Goldreich et al. (2019). In particular, staphylococci are the most common gram-positive bacteria secreted during keratitis. Second-generation fluoroquinolones, ciprofloxacin, aminoglycosides, and gentamicin have been found to be highly effective against most isolates (Lin & Petersen-Jones, 2008). According to our research, conjunctival staphylococci are sensitive to most antibiotics. In addition, staphylococci also account for the largest number of microorganisms detected.
Conjunctivitis in cats has a different etiology, which is confirmed by the analysis of reports from other researchers. In particular, in cats with chronic conjunctivitis DNA of C. felis and FHV-1 was found in 6.7% and 33.3% of patients, respectively, despite the absence of joint infections with both pathogens (Wieliczko & Ploneczka-Janeczko, 2010). Chlamydia and Mycoplasma are an important cause of acute and chronic conjunctivitis in cats (Sykes, 2005; Low et al., 2007).
The assumptions about the polyetiological nature of conjunctivitis in cats are supported by studies by other authors. In particular, cat conjunctivitis is often associated with herpes viruses, mycoplasmas, and chlamydia (Sjodahl-Essen et al., 2008). The authors point out that the presence of these pathogens may not be associated with the primary disease, but is a consequence of the weakening of the body's defenses through the primary disease, or may be the cause of the primary disease. The authors were unable to establish a correlation between the positive results of the diagnostic tests and the degree of clinical manifestation. It should be noted that by polymerase chain reaction smears of the conjunctiva of clinically healthy cats, we also isolated C. psittaci and M. felis. But it is important to note that initial diagnosis should not be based solely on laboratory testing. Researchers indicate that the presence of C. felis is associated with conjunctivitis, whereas the detection of the herpes virus does not correlate significantly with the clinical signs of the disease. This suggests that the polymerase chain reaction alone does not provide an accurate diagnosis of conjunctivitis associated with the herpes virus (Rampazzo et al., 2008). A large percentage of chlamydial conjunctivitis in cats was determined by our studies.
According to Aftab et al. (2019), conjunctivitis is not characterized by seasonality against the background of predominance of gram-positive bacteria - S. epidermidis (43.2%), P-hemolytic streptococcus (18.9%), S. aureus (17.9%) and E. coli (11.5%). At the same time, we found that their registration in the cold season was significantly higher, although gram-positive microorganisms were most often also isolated. This may be due to the fact that, in addition to bacterial microflora associations, the level of morbidity is also influenced by other factors: anatomical disorders, traumas and hypersensitivity reactions, either alone or together with infectious factors.
With only information on the colonization of the conjunctival sac of S. aureus, we have established the peculiarities of its distribution in tissues and fluids of the eye: the highest concentration is established in
the cornea and conjunctiva against the background of much lower concentration in the chamber fluid and blood, which can be explained by the activation of protective mechanisms, given the ability of S. aureus to cause intraocular inflammation and loss of retinal function (Kumar & Kumar, 2015). The activation of the conjunctivitis complement system in cats, which is caused by S. aureus, has been proven to be consistent with the message of other researchers, including Gilger (2008). Activation of the complement system is controlled by regulatory proteins that determine its intensity, sufficient to destroy the pathogenic factor (Sohn et al., 2000). Frequent prolonged exposure to systemic or local inflammatory stimuli may result in low levels of complement activation and process generalization (Crowley et al., 2018). The results of the dynamic profile of complement activation are valuable for a better understanding of the mechanism of conjunctivitis in cats and the development of pathogenetically sound treatment guidelines.
Despite the important role of neutrophils in the mechanisms of innate immunity, we have not established significant changes in their blood content for conjunctivitis caused by S. aureus, which is probably related to the ability of S. aureus to produce protection factors by escaping recognition (McGuinness et al., 2016; Rasigade, 2018).
In most cases, the inflammatory response is accompanied by a decrease in the level of acute-phase proteins by Ceron et al. (2008), however, we did not detect significant changes in albumin content due to purulent conjunctivitis caused by S. aureus.
The established enhancement of both cellular and humoral defense mechanisms is explained by the activation of factors of the innate immune response (Yoong & Pier, 2010).
Analyzing the results obtained, it can be stated that eye microflora in normal and during pathological conditions is an important aspect of the evaluation of eye diseases. During the examination of the microbial flora, it was found that gram-positive bacteria are predominant in the conjunctival cavity both in norms and pathologies. Injuries to the eyes can lead to reproduction and potential pathogenicity of the normal microflora of the eye, which is consistent with our findings. The diversity of bacterial, fungal, viral and other microbial isolates influences the diagnosis and treatment of eye diseases (Paul & Gerding, 1990).
The results obtained may be useful in the diagnosis and treatment of conjunctivitis, as certain types of staphylococci may have different mechanisms of pathogenicity, pathogenesis, or transmission features. The authors suggest that S. aureus is a natural bacterial flora in cats, especially in animals that are kept in close contact with their owners (Na-gase et al., 2002). In the group of healthy cats kept in households, a greater variety of staphylococcus species was observed than in wild cats. Researchers point to the fact that conditionally pathogenic microflora can cause a wide range of complications and report that the morbidity, antibiotic resistance of different types of staphylococci in cats depends on the state of the body's defenses. A statistically significant correlation was observed between cat health and staph infection (Biero-wiec et al., 2019). The high frequency of colonization of cats by staphylococci is confirmed by Ma et al. (2019), who isolated them in 73.8% of animals, which is consistent with the results of our studies. Antibiotic sensitivity in different types of staphylococci varies. In animals, the pathogenic potential of these microorganisms is not yet fully understood (Gandolfi-Decristophoris et al., 2013).
Of particular concern to scientists in the field of public and animal health is the methicillin-resistant S. aureus. In this regard, the importance of providing effective and reliable methods for its identification is emphasized (Medhus et al., 2013; Morris et al., 2006), which in the long run confirms the need for research to investigate the role of staphylo-cocci in the incidence of conjunctivitis and keratitis in cats.
The findings are consistent with the concept of the Working Group of the International Society for Infectious Diseases of Companion Animals, which substantiates the feasibility of further studies of bacterial diseases of cats to develop more effective recommendations for their treatment and prevention (Lappin et al., 2017).
Conclusions
Cat diseases are very common, with a large proportion of conjunctivitis (35.4%) and keratoconjunctivitis (29.2%), most of which were reported in the cold season, and were caused by mechanical trauma, coccal and chlamydial infections. Among the detected microorganisms, the vast majority (81.9%) were staphylococci, including S. albus, S. aureus, S. epidermidis. In spite of the high degree of sensitivity of the detected microflora to the most common antibiotics in veterinary practice, we consider it logical to assume that this microflora is normal and conditionally pathogenic for the conjunctiva. Features of antigen distribution in blood, tissues, and eyeball fluid in staphylococcal keratoconjunctivitis may be evidence of the ability of staphylococci to ignore cellular protective barriers, although the highest antigen concentration is found in the cornea and conjunctiva. Dynamics of significant increase in the content of circulating immune complexes, immunoglobulins, powerful activation ofphagocyto-sis and the complement system in the classical way are evidence of distinct cellular and humoral reactions to the infectious agent.
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