Feline herpesvirus 1 viral load related to environmental factors in sheltered Feline herpesvirus 1 viral load related to environmental factors in sheltered cats

: Shelter environment stress factors are related to FHV-1 viral reactivation. However, comparisons between conjunctival viral load and environmental factors have not been commonly evaluated. The aim of this study was to correlate FHV-1 viral load in domestic cats with and without clinical signs of conjunctivitis to shelter design in order to use FHV-1 viral load as a parameter of “health management”. Cats from four different shelters underwent an ophthalmological examination. Samples were collected by rolling a DNA/RNAse-free cytobrush over the ventral conjunctival fornix and were stored in 1.5 mL sterile microtubes in 500 μ L of Eagle’s minimum essential medium and kept at 4 ºC. Molecular procedures were performed up to 48 hours after collection. Different routines regarding new arrivals were directly related to FHV-1 viral load. Shelters where new arrivals occurred on daily basis had the highest viral load (2.69x10 8 copies/µL), while those shelters where new arrivals had not occurred in the few months prior to the beginning of the study had the lowest rate (1.63x10 3 copies/µL). Environmental factors directly influenced FHV-1 DNA viral load. This study highlighted the need to improve the management approach in the animal shelter environment to reduce stressful situations responsible for FHV-1 reactivation and higher viral load quantification.


INTRODUCTION
Animal shelters house a large number of animals from different species, have a high turnover of animals, high population density, and a mixture of cats from different source colonies (FINKA et al., 2014;. These characteristics have long been recognized as crucially important determinants for the risk of acquiring contagious infectious diseases (TANAKA et al., 2012;PESAVENTO & MURPHY, 2014). Domestic cats are common sheltered animals and conjunctivitis is one of the most frequent diseases at sites with large population concentrations (DAVIS-WURZLER, 2014;. Feline herpesvirus 1 (FHV-1) is one of the pathogens responsible for conjunctivitis (BAUMWORCEL et al., 2017;FERNANDEZ et al., 2017) FCV, Mycoplasma felis, and Chlamydophila felis. However, infected animals establish latency;, ;therefore, the first infection may result in reactivation in stressful situations (MÖSTL et al., 2013). The Baumworcel et al. particular conditions to which these animals are submitted in shelters generate a stress situation that facilitates the reactivation of FHV-1 and; consequently, the spread of infection to susceptible animals (STELLA & CRONEY, 2016).
For these reasons, understanding the importance of stress in shelter cats and being able to identify and mitigate stress whenever possible is critical for maintaining healthy shelter cat populations (AMAT et al., 2016). Thus, determining which shelter characteristics influence FHV-1 viral load is essential for improving the design and management of shelters as well as kitten welfare. The characteristics analyzed in the present study were parameters previous established by the Association of Shelters Veterinarians and included building design adaptation, temperature control, presence of nonporous surfaces, proximity to other species, mode of housing animal (caged or not), distance between cages (>45cm), minimal spacing (60 cm) between litterbox, resting place and food, and presence of an in-house veterinarian.
The aim of this study was to correlate FHV-1 viral load in domestic cats with shelter design (shelter environmental factors, animal housing characteristics, and presence of an in-house veterinarian) in order to use FHV-1 viral load as a parameter of "health management".

Study locations -shelters
Shelter enrollment in this study was convenience based and criteria for inclusion were permission from the shelter manager, ability to collect required data, and distance from the shelter and laboratory not exceeding 70 kilometers. Four different shelters in Rio de Janeiro, Brazil were included in this study. Shelters were designated as A, B, C, and D according to the order in which they were visited at the beginning of the study. In all four shelters, cleaning methods were similar, and they all used benzalkonium chloride 2%. Each shelter had its own responsible technical team.
New arrivals occured differently in each shelter. In shelter A there were no arrivals in prior months to our visit. In shelter B, new arrivals occured on weekly basis. In shelter C new arrivals occured on daily basis while in shelter D new arrivals occured randomly. The animal housing characteristics and shelter environmental factors are listed in table 1.

Study population
This research was conducted with UFF Ethics Committee of Animal Research approval (CEUA Nº 330/2013 and CEUA N º 708/2016). A total of 70 intact kittens (28 males and 42 females) underwent an ophthalmological examination by the same veterinarian (NB) who swabbed all cats from this study between September 2015 and September 2016. The inclusion criteria were: kittens up to 12 months old without previous use of systemic or topic ocular drugs. All kittens from each one of the four shelters visited that matched the inclusion criteria were included. Topical anesthetics and fluorescein staining were not used during the ophthalmological examination because these compounds can affect the sensitivity of qPCR methods (GOULD, 2011;HORZINEK et al., 2013). A defined set of criteria for the conjunctivitis score system was adapted from a previously established one (HARTMANN et al., 2010) and was used in all shelters. Score 0 standed for no clinical signs of conjunctivitis, score 1 for mild conjunctival hyperaemia, score 2 for moderate conjunctival hyperaemia and mild chemosis, while score 3 for severe conjunctival hyperaemia and moderate to severe chemosis. All animals housed in the same shelter that matched inclusion criterias were added to this study. Cats without conjunctivitis were swabbed first, but at the same visit as those with conjunctivitis. A total of 41 kittens had clinical signs of conjunctivitis. Twenty-nine kittens without clinical signs of conjunctivitis and without any previous history of ocular disease with the same age as those with clinical signs were also swabbed. In shelter A there were 14 cats in total but only eight of them matched the inclusion criteria. In shelter B, there were 22 cats in total but only 17 of them matched the inclusion criteria. In shelter C, there were around 150 cats in total, but only 31 of them were in an accessible area and matched the inclusion criteria. For last, in shelter D there were 24 cats in total, 14 of them matching the inclusion criteria.

DNA extraction and quantitative PCR (qPCR)
From the conjunctival samples, a pool was made, combining the right eye sample with the left eye sample from the same animal. Aliquots were vortexed (10.000X per 5 min) to assure consistente conditions for DNA extractions and subsequente analysis of FHV-1. The DNA extraction was performed using a PureLink spin column-based kit for genomic DNA (Invitrogen) according to the manufacturer's instructions. qPCR was performed to detect and quantify the FHV-1 target gene TK, according to previously described conditions (HELPS et al., 2003). A cycle threshold (Ct) <38 was considered to indicate a clear positive result and it was standardized automatically by StepOne™. (Applied Biosystems). All tests included a negative control containing Milli-Q water. The Felocell CVR-C (Zoetis) vaccine was used as the positive control.
Briefly, the 25-µL real-time PCR reaction contained 12.5 µL of a TaqMan ® Universal Master Mix ll with UNG ; 1.0 µL (10 µM) of each primer (Primer F -GGACAGCATAAAAGCGATTG; Primer R -AACGTGAACAACGACGCAG) with 74bp ; 0.5 µL (µM) of probee (FAM-QSY); 5.0µL of sterile water; and 10 µL of extracted template DNA. All reactions were performed on a StepOne (Applied Biosystems™) thermocycler for the following conditions: 2 minutes at 50 o C, 10 minutes at 95 o C, and then 40 cycles (each cycle consisted of a denaturation step [15 seconds at 95 o C] followed by an annealing step [1 minute at 60 o C]).
Realtime test was standardized according to MIQE guidelines (BUSTIN et al., 2009). All samples, standard curve dilutions positive and negative controls were tested in duplicates. The method used to quantify FHV-1 DNA viral load was using a synthetic curve as standard curve.
The synthetic curve was drawn through GeneArt program. The gene block was synthesized on a 2448bp plasmid DNA segment with a 89bp insert. The sequence that encodes for TK gene and used as template was available from GenBank (JX628812).
For dilution of the synthetic standard curve, the 5 µg of lyophilized plamidial DNA were diluted in 200 µL of TRIS EDTA solution quantified at (0.016 ng/µL). The concentration reported in ng/ µL was transformed into genome copy numener per microliter. Serial dilutions were made at base 10, for the standard qPCR curve. The Ct value of each dilution was then compared to target copy number in order to establish a standard curve to be used. Dilutions were 10 0 -10 8 copies/µL.

Statistical analyses
The mean values of viral load were initially converted to log 10 in order to decrease the variation as a function of the mean and to obtain a normal distribution. Analysis of variance (ANOVA) was used to compare mean viral loads in animals with and without clinical signs of conjunctivitis and to compare shelter characteristics with viral loads. P values <0.05 were considered statistically significant.

RESULTS
Animals were 2-10 months old (mean age of 4.7 months ± 2.7 months). The FHV-1 DNA was detected in equal prevalence regardless of gender, in all shelters; although, some characteristics had more influence on viral load than others. Not all parameters established by the Association of Shelters Veterinarians (ASV) and analyzed in this study, were observed at four shelters. Parameters analyzed according to FHV-1 viral loads are summarized in table 2. Mean viral load according to new arrivals system is listed in table 3.
In shelter A all eight animals tested positive in qPCR assay for FHV-1 DNA. In shelter B out of 17 animals tested, only two of them were negative. In shelter C, out of 31 only one was negative while in shelter D, four out of 14 were negative.

DISCUSSION
The prevention of infectious disease is one of the main objectives of shelter veterinary medicine. To reduce FHV-1 infection, it is important to determine the potential risk factors that increase FHV-1 DNA viral load in sheltered cats.
This was the first study to quantify FHV-1 DNA viral load in cats from different shelters in Brazil. The prevalence of FHV-1 in cats from shelters was determined as being up to 90% using the qPCR assay. The qPCR assay used Thymidine Kinase (TK) gene as the target gene. The TK is part of the kinases family of enzimes with high degree of conservation between species and inside the same specie (SOLAROLI et al., 2006) feline herpesvirus TK (FHV-TK. The use of this kind or target gene reduces the incidence of false results.
The detection of an extremely high number of cats positive for FHV-1 was a surprise since a previous study in a similar population using PCR detected FHV-1 DNA in 57.4% of samples (BAUMWORCEL et al., 2017)FCV, Mycoplasma felis, and Chlamydophila felis. This difference could be explained by the sensitivity of the qPCR technique (LITSTER et al., 2015)for a total of 22 study cats. Combined conjunctival and oropharyngeal swab specimens were tested by quantitative real-time PCR (qPCR. There was no difference on FHV-1 viral load according to sex. The detection of FHV-1 regardless of gender confirms previous studies (GRAHAM;MAAZI et al., 2016).
A statistical difference in detection FHV-1 DNA between animals with and without clinical signs of conjunctivitis was observed. However, a minimum infectious dose has not been determined for FHV-1 infection, any viral load can be considered potentially relevant. The high level of positive asymptomatic animals indicated that they cannot be excluded from suspected FHV-1 infection. Clinical screening alone is insufficient for appropriate control of FHV-1 infection in shelter kittens and qPCR should be used as a diagnostic tool. Nevertheless, it is not possible to rule out that potential confounding between health status and arrival time may have introduced bias into this analysis.
Shelter spaces tend not to be completely welcoming, and a minimum level of stress is inevitable. Feline herpesvirus is directly reactivated by stress (GOURKOW & PHILLIPS, 2015) and is a common cause of infectious diseases in shelters. To understand the importance of stress in shelter cats and be able to identify and mitigate Table 2 -Shelter characteristics including environmental factors, animal housing characteristics and presence of an in-house veterinarian and their respective statistical significance in relation to FHV-1 DNA viral load. All kittens from each one of the four shelters visited that matched the inclusion criteria were included in mean viral load (copies/µL) calculation.

SD -Standard Deviation
stress whenever possible is critical for maintaining healthy shelter cat populations (AMAT et al., 2016). Thus, to be able to point out which shelter characteristics influence FHV-1 viral load is essential for improving the design and management of shelters as well as kitten welfare.
Our results revealed that environmental risk factors including building design adaptation, temperature control, and presence of non-porous surfaces cannot be considered separately. Shelters that were not originally built to be shelters had higher viral load than those that were planned and built as shelters. However, decreasing population density in adapted shelters could contribute to decrease stress and, consequently, lower viral loads. A smaller number of animals could contribute to better quality housing, as previously suggested regarding capacity for care (C4C) (KARSTEN et al., 2017). The frequent introduction of new animals might be an explanation for virus reactivation. Different routines regarding new arrivals were directly related to FHV-1 viral load. Shelters where new arrivals occurred more frequently had the highest viral load. A bias of this study might be when after shelter arrival samples were collected, as this could impact level of stress and consequently increase FHV-1 DNA viral load.
Although, a minimum distance of 45 cm between cages has been stipulated by the ASV (NEWBURY et al., 2010), this environmental factor did not contribute to higher viral loads when this distance was not observed. The lack of difference could be explained by the distance viral load particles migh travel in sneezes, that varies from 1-2 meters (POVEY; JOHNSON, 1970). The distance between cages probably has to be greater than one meter for this factor to have an effect. Our study suggested that caged animals have higher viral loads than uncaged ones. Uncaged animals have a better quality of life than caged ones (WAGNER et al., 2018). Being caged is a stressful factor and could reflect the recrudescence of latent FHV-1 infection as a result of stress.
The proximity to other animal species was related to higher viral loads reported on shelters B and C. Noises and odors from other species is not well tolerated by sheltered animais (STELLA; CRONEY, 2016)free-roaming (70 million. It increases stress in cats (NEWBURY et al., 2010). Our results reinforce the necessity for an appropriate acoustic environment for good animal health and welfare.
The presence of an in-house veterinarian did not contribute to lower viral loads. An effective FHV-1 infection control program requires more than solely veterinary procedures. Non-medical factors, such as the architectural design of the shelter and an appropriate housing system should also be considered in addition to an in-house veterinarian, as previously discussed (FINKA et al., 2014).
Some stress-related issues can be solved, and others reduced. In one way or another, this approach may decrease FHV-1 reactivation and viral load quantification. The information provided here; although, a bigger sample data would be more representative, may be extrapolated to other shelters and may help to improve the management approach in animal shelters. In this manner, it could reduce stress situations that can contribute to FHV-1 reactivation. It is important to emphasize that there is no standard protocol to be adopted generally by all shelters. It should be adjusted to each shelter budget and structural conditions.

CONCLUSION
All environmental stress factors analyzed in this stu dy, except for the distance between cages, were directly related to higher viral loads of FHV-1. This study highlighted the need to improve the management approach in the animal shelter C (daily basis) +/-150 31 2,69x10 8 ± 1,04x10 9 D (randomly) 24 14 7,03x10 6 ± 2,61x10 7 SD -Standard Deviation. environment in order to reduce stress situations responsible for FHV-1 reactivation and higher viral load quantification.