Introduction

Respiratory illness is a common reason for cats to visit a veterinary clinic¹ and euthanasia in some shelters.² Infectious agents are often the cause of feline respiratory diseases. Two viruses, feline calicivirus (FCV) and feline herpesvirus-1 (FHV), both cause coughing, sneezing and ocular/nasal discharge, but FCV can also cause gingivitis/stomatitis or oral ulcers.^3–5 Bacterial organisms such as Chlamydia felis, Bordetella bronchiseptica and Mycoplasma species can cause ocular/nasal discharge, sneezing and conjunctivitis.^3,6–10 While C felis is more likely to cause ocular signs than respiratory illness,⁶ B bronchiseptica causes bronchopneumonia and cough, although cough is less common in cats than dogs.^3,9,10 A highly pathogenic influenza A virus such as H5N1 can induce high fever, ataxia, circling and high mortality,¹¹ and a low pathogenic influenza virus originating from humans, dogs or birds causes cough, sneezing and ocular/nasal discharge with more serious signs often attributed to secondary bacterial infections.¹² Further testing is often required to differentiate which pathogen is present in feline ocular and respiratory infections. Diagnosis and treatment are often presumed, as additional tests can be costly and time-consuming.

PCR testing has largely replaced bacterial culture and virus isolation due to higher sensitivity and quicker turnaround times.¹³ Georgia Veterinary Diagnostic Laboratories (GVDLs) provide two options for PCR panels: the ocular panel targeting FHV, FCV and C felis, and respiratory panel detecting FHV, FCV, C felis, Mycoplasma species, B bronchiseptica and influenza A virus. This study aimed to summarize the findings of the ocular and respiratory panels from clinical cases submitted to GVDLs, including infectious agents detected in the upper and lower respiratory tracts.

Materials and methods

Real-time PCRs

The real-time PCR methods have been previously described for the targets in the ocular and respiratory panels including FHV,¹⁴ FCV,^15,16 C felis¹⁴/Chlamydia species¹⁷ and Mycoplasma species,¹⁸ B bronchiseptica¹⁹ and influenza A virus.²⁰ Primer and probe sequences are listed in Table 1. C_t values lower than 35 or 40 are defined as positive detection. All PCR methods were conducted with proper controls to the standards of the American Association of Veterinary Laboratory Diagnosticians.

Table 1

Primer/probe used in the ocular and respiratory panels at Georgia Veterinary Diagnostic Laboratories

Data analysis

Excel spreadsheets (Microsoft) were used to analyze pathogen detections and locations in each age group (defined according to the American Association of Feline Practitioners and American Animal Hospital Association charts, with ages rounded to the nearest group) (Table 2). Specimens taken from below the pharynx are defined as lower respiratory samples, such as transtracheal wash and lung tissue. Pathogen detection between age groups was compared by Fisher’s exact test and correlation matrix by Spearman’s correlation in Prism, version 10 (GraphPad Software).

Table 2

Age groups and the detection numbers by ocular and respiratory panels

Results

Between 2012 and 2022, 120 submissions were received for ocular panel. Ocular sites such as conjunctival/corneal swab and ocular fluid comprised 56.7% (68/120) of the specimens and 17.5% (21/120) were from mixed locations including an ocular site (eg, nasal/conjunctival swab). The remaining 31 specimens were taken from upper respiratory areas such as nasal/oral or undefined sites.

The most common detection was FCV (28.8% [17/59]), followed by FHV (25.4% [15/59]) and C felis (20.3% [12/59]). Fifteen cases (25.4%) had more than one pathogen detected, including FCV + FHV (10.2% [6/59]), FCV + C felis (6.8% [4/59]), FHV + C felis (5.1% [3/59]) and FCV + FHV + C felis (3.4% [2/59]) (Figure 1). Pathogens were analyzed to examine if certain agents tended to be detected together. There was no significant correlation between the pathogens (P >0.05).

Figure 1

Number of detections by feline ocular PCR panel at Georgia Veterinary Diagnostic Laboratories from 2012 to 2022. Panel targets include feline herpesvirus-1 (FHV), feline calicivirus (FCV) and Chlamydia felis/Chlamydia species In total, 59 cases had at least one organism detected from 120 submissions (49.2% positivity rate)

Cats aged younger than 3 years made up 59.3% (32/54) of the cases (Table 2). While 47.1% (8/17) of FCV detections were from non-ocular sites (such as nasal/oral/lung), most single detections of C felis (91.7%, 11/12) and FHV (80.0%, 12/15) were from ocular specimens.

Between 2012 and 2022, 453 submissions were received for respiratory panel. Overall, the panel showed 30.7% (139/453), 14.6% (66/453), 2.4% (11/453), 51.9% (235/453), 3.5% (16/453) and 0.2% (1/453) positivity rate for FCV, FHV, C felis, Mycoplasma species, B bronchiseptica and influenza A virus, respectively. Most specimens (82.3%, 373/453) were taken from respiratory sites such as nasal/oral/tracheal swab, nasal flush/tissue and lung tissue, 3.09% (14/453) of samples were from ocular sites and 5.96% (27/453) were from mixed locations including an ocular site (eg, oral/nasal/conjunctival swab). The remaining 8.61% (39/453) specimens were taken from other or undefined sites.

The most common single detection was Mycoplasma species (36.9%, 116/314), followed by FCV (13.7%, 43/314) and FHV (7.6%, 24/314) (Figure 2). Multipathogens comprised 127 cases (40.4%), including FCV + Mycoplasma species (20.4%, 64/314), FHV + Mycoplasma species (5.41% 17/314) and FHV + FCV + Mycoplasma species (4.78%, 15/314) (Figure 2). No cases tested positive for more than four pathogens (Figure 2). A significant correlation was identified between co-detection of FCV + Mycoplasma species (P = 0.01), C felis + Mycoplasma species (P = 0.044) and Bordetella species + Mycoplasma species (P = 0.004) (Figure 3).

Figure 2

Number of detections by feline respiratory PCR panel at Georgia Veterinary Diagnostic Laboratories from 2012 to 2022. Panel targets include feline herpesvirus-1 (FHV), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica, Mycoplasma species and influenza A virus. In total, 314 cases had at least one organism detected from 453 submissions (69.3% positivity rate)

Figure 3

Pathogen detection correlation matrix by feline respiratory PCR panel. Number 0 or 1 was assigned for negative or positive results, respectively, for conducting the correlation coefficient analysis. Spearman’s r is shown for each pair of pathogens. Four pathogens showed significant correlation of co-detection including FCV + Mycoplasma species (P = 0.001), Chlamydia felis + Mycoplasma species (P = 0.044) and Bordetella species + Mycoplasma species (P = 0.004). FCV = feline calicivirus; FHV = feline herpesvirus-1

Age distributions showed that most infections were among junior animals, followed by kittens and prime (25.5%, 21.8% and 18.9%, respectively) (Table 2). The most common pathogens found in cats aged 10 years and under were Mycoplasma species, FCV + Mycoplasma species and FCV (Figure 4). For cats younger than 7 years old (kitten + junior + prime), FHV detections were significantly less than those of other age groups (P = 0.0099), while FCV + Mycoplasma was significantly less in senior and geriatric cats than in younger age groups (P = 0.042) (Figure 4).

Figure 4

Pathogen detection percentage by feline respiratory PCR panel. Of the 314 positive cases, 275 had age provided. The kitten, junior, prime and mature age groups all had feline calicivirus (FCV), Mycoplasma species and FCV + Mycoplasma species as the most common pathogens. The senior age group had Mycoplasma species, feline herpesvirus-1 (FHV) and FCV as the most common pathogens. The geriatric age group had Mycoplasma species and a tie between FHV + Mycoplasma species and FCV.

*Coinfection of FCV + Mycoplasma species is significantly less common in senior and geriatric cats than in younger cats (P = 0.042);

**single FHV infection is significantly less common in cats younger than 7 years (kitten + junior + prime) than in cats older than 7 years (mature + senior + geriatric) (P = 0.0099)

While most detections were from respiratory specimens (81.8%, 257/314), the majority of the triple detection of C felis + FCV + Mycoplasma species (80%, 4/5) were from mixed sites including an ocular site (eg, oral/nasal/conjunctival swab). The only positive specimen identified for influenza A virus was lung tissue. Based on the recorded physical locations, specimens from the respiratory panel were further divided into upper vs lower respiratory tract. Positivity rates were slightly higher in upper respiratory samples (Table 3). While single pathogen cases were higher in upper respiratory samples, multipathogen detections were higher in lower respiratory samples (Table 3). The top three detections were the same for upper vs lower respiratory tract but in different order: Mycoplasma species (39.6%, 97/245), FCV + Mycoplasma species (19.2%, 47/245) and FCV (12.7%, 31/245) vs FCV + Mycoplasma species (25%, 10/40) and a tie between Mycoplasma species and FCV (22.5%, 9/40), respectively (Figures 5 and 6). While no case had four or more pathogens in the upper respiratory tract, one case of FHV + FCV + Bordetella species + Mycoplasma species was identified in the lower respiratory tract (lung tissue) (Figures 5 and 6).

Table 3

Detection numbers of specimens taken from upper vs lower respiratory tract* by the respiratory panel

Figure 5

Number of detections from the upper respiratory tract by feline respiratory PCR panel. Among specimens taken from upper respiratory tract, 69.8% (245/351) tested positive with the three most common pathogens being Mycoplasma species, FCV + Mycoplasma species and FCV. FCV = feline calicivirus; FHV = feline herpesvirus-1

Figure 6

Number of detections from the lower respiratory tract by feline respiratory PCR panel. Among specimens taken from lower respiratory tract, 65.6% (40/61) tested positive with the three most common pathogens being FCV + Mycoplasma, Mycoplasma species and FCV. FCV = feline calicivirus; FHV = feline herpesvirus-1

Discussion

Prevalence studies for feline ocular and upper respiratory infections have been reported from the UK,²¹ Germany,²² Spain²³ and Australia,²⁴ as well as for shelter animals in Korea,²⁵ Canada²⁶ and the USA.^13,27,28 However, retrospective data have not been reported from a diagnostic laboratory in the USA. To give regional veterinarians a better idea of the pathogens detected in feline ocular and respiratory infections, we summarized the 2012–2022 results from GVDLs’ PCR panels.

This study included samples submitted by veterinarians and was not exclusively obtained from shelter populations. Our findings are consistent with those investigating general populations, with FCV and Mycoplasma species being the two most detected pathogens.^23,24 On the other hand, studies focusing on shelter cats mostly found FHV or Mycoplasma species to be the most detected pathogen.^13,25–27

Not all submissions for the ocular panel comprise only ocular specimens. While some veterinarians may have chosen the ocular panel over the respiratory panel for economic reasons, others may have taken swabs from multiple respiratory sites to increase the chance of pathogen detection. After all, respiratory tract infections often involve signs in both ocular and respiratory systems.^3–10,12

Interestingly, Mycoplasma species comprise most detections of both upper and lower respiratory submissions. Although, Mycoplasma species are considered to be a commensal of the upper airway and conjunctiva of cats, a weak but significant association was found between Mycoplasma species and upper respiratory tract disease in a meta-analysis study.²⁹ While it is not conclusive that Mycoplasma species are a primary pathogen of the upper respiratory tract in the current study, the fact that it is the only detected agent in most upper respiratory disease cases does provide some evidence to support this argument. Yet, the detection of a pathogen does not mean it is solely responsible for causing the disease, as other pathogens could have been missed if early in the course or other etiologies such as asthma or neoplasia could also be involved predisposing to mycoplasmal infection.⁸ Mycoplasma species detection was found to be significantly correlated between FCV, C felis and B bronchiseptica (Figure 3), suggesting the role of secondary infection in those cases.

FCV was detected more often than FHV in the current study; however, FHV infection leads to latent infection after recovery, and thus, the prevalence of FHV could be underestimated. While 23.9% (75/314) cases submitted for the respiratory panel had viral-only detections, about 76.1% (239/314) were bacterial-only or had bacterial components, suggesting that empirical treatment with antibiotics may have merit. B bronchiseptica and influenza A virus were detected in very few cases, suggesting they are not main contributors to feline respiratory cases submitted to our laboratories in Georgia.

Kittens and junior cats (<3 years old) seemed to make up most cases received in both diagnostic laboratories and had the most mixed infections. Although low in number, mature and senior cats had a larger percentage of FHV infections (Figure 4), possibly due to latent FHV recrudescing in older animals. Mycoplasma species had the most frequent detection in all age groups, except in kittens where it was the second most common.

Due to the limitation of the data source, we did not have complete histories for most submitted cases. Lack of information on treatment, housing conditions and clinical signs hampered our ability to draw stronger conclusions from our data. Nevertheless, it is fair to assume that animals submitted for the ocular and respiratory PCR panels were showing signs consistent with infection of either ocular, respiratory or both. Given this, it is reasonable to conclude that positive detections by the PCR panels are likely to be at least contributing to the disease state. Another limitation of the current study is that PCR results cannot rule out pathogens not included in the panel such as Staphylococcus and Streptococcus species.

Conclusions

FCV and FHV were the most frequent detections by the ocular panel, with Mycoplasma species and FCV being the most frequent detections by the respiratory PCR panel. As many cases involved FCV and/or FHV, incorporating FCV and FHV in vaccination regimens could help reduce the number of cases and/or lessen the clinical signs. Mycoplasma species were the major pathogen detected in both upper and lower respiratory tracts. Despite its controversial role as a primary pathogen, there is value in considering antibiotic treatment for Mycoplasma species in feline respiratory cases.