Seroprevalence of feline immunodeficiency virus,feline leukaemia virus and Toxoplasma gondii in stray cat colonies in northern Italy and correlation with clinical and laboratory data

Abstract

Stray cat colonies in urban and rural areas of Lombardy, northern Italy, were surveyed for seroprevalence of feline immunodeficiency virus (FIV) antibodies, feline leukaemia virus (FeLV) antigen and Toxoplasma gondii IgG. Of 316 cats tested, 6.6% were positive for FIV and 3.8% were positive for FeLV infection; 203 cats were tested for T gondii IgG antibodies and a prevalence of 30.5% was detected. Statistical analysis tested the influence of provenience, age, gender, health status and laboratory results on seroprevalence and found male gender and adult age were risk factors for FIV infection. FIV-infected cats were more likely to have a decreased red blood cell count than FIV seronegative cats. No predictors were significantly associated with FeLV and T gondii seropositivity. Colony cats in this study posed a limited risk for retrovirus infection to pet cats allowed outdoors, whereas toxoplasmosis exposure was comparable with the worldwide data.

Introduction

Stray cats may serve as reservoirs of infectious agents for domesticated pet cats, such as feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV), which are important causes of morbidity and mortality in cats.¹ Both these retroviruses have a worldwide distribution. FeLV is transmitted primarily through the sharing of food and drink containers and by behaviour such as social grooming. FIV is transmitted mostly through bite wounds, but can also be transmitted in utero or through colostrum and milk.^2,3 FIV and FeLV prevalence varies widely depending on lifestyle, gender, health conditions and other variables of the cat populations examined.^4-13 The American Association of Feline Practitioners and the European Advisory Board on Cat Diseases recommend that the retrovirus status of all cats should be known.^1,14,15 Knowledge of the prevalence of FeLV and FIV infection in stray cat colonies is important to define effective prophylactic and management programmes for stray feline populations to guarantee the welfare of the cats themselves and to minimise the threat posed to pet cats with outside access.

Stray cat populations may also play an important role in the transmission of several pathogenic agents to humans. Of the zoonotic agents transmitted by cats, T gondii, is the most relevant feline parasite. Although transmission of this enteric coccidian parasite can occur following ingestion of undercooked meat, T gondii oocysts pass into the environment from infected cats and are a significant source of infection for humans and other mammals. Cats are the only source of T gondii oocysts in urban environments and information on the prevalence of exposure in cats is useful for assessing environmental contamination by the protozoan.¹⁶

In northern Italy, no recently published data exist for FIV, FeLV or T gondii infection in a large population of stray cats from colonies. The objective of our study was to determine seroprevalence of FIV and FeLV infections and T gondii exposure among stray cat colonies in urban and rural areas in Lombardy, northern Italy. The correlation of seropositivity with laboratory data and parameters such as origin, gender, age and health status were also examined and compared between infected and uninfected cats.

Materials and methods

Sampling and data collection

Blood samples and individual animal data were collected from 316 European shorthair stray cats from urban and rural colonies located in the Lombardy region of northern Italy. These samples were collected by veterinary surgeons at the Faculty of Veterinary Medicine through participation in the trap-neuter-release (TNR) programme between January 2008 and January 2010. Cats were trapped by volunteers and delivered to the Department of Clinical Sciences at the University of Milan. Cats were anaesthetised with a combination of tiletamine and zolazepam (Zoletil 100; Virbac, Milan, Italy) at a dose of 12 mg/kg plus tramadol (Altadol; Formevet, Italy) 1 mg/kg, given intramuscularly in the thigh muscles, based on estimated body weight, while cats were confined in the trap. General anaesthesia was maintained with isoflurane (Isoflo; Esteve, Milan, Italy) given by mask. Cats’ eyes were protected with an ophthalmic lubricant during anaesthesia to prevent drying of the cornea. At the end of the surgery, 1 cm of the distal tip of the left ear was removed by placing a haemostat proximal to this position and cutting off the tip with surgical scissors. This permanent mark identified sterilised cats and prevented them from being represented for surgery. Age (estimated based on dentition — animals ≤6 months of age were considered juvenile, whereas all others were considered adult), gender (male or female) and origin (urban or rural) of each cat were recorded together with data obtained from physical examination of the cats (healthy or unhealthy). Unhealthy cats were defined as cats with the presence of one or more of the following clinical abnormalities: lymph node enlargement, pale mucous membranes, stomatitis, or signs of ocular and respiratory infections. Blood samples were collected aseptically from the jugular vein during anaesthesia and placed in EDTA-treated tubes and in serum separator tubes. Cats were hospitalised for five or more days after the surgery depending on health status and then released to the location where they were trapped. All the procedures were approved by the local authority of the city council.

Within 24 h of sample collection, a complete blood count (CBC) was performed on whole blood using an ADVIA 120 System (Siemens Healthcare Diagnostics, Milan, Italy). Cats were categorised in terms of presence or absence of anaemia [defined as decreased packed cell volume (PCV) and/or red blood cell (RBC) count and/or haemoglobin (Hb)], leukopenia, leukocytosis, neutrophilia, neutropenia, lymphocytosis and lymphopenia. Following separation, sera were tested for antibodies to FIV (relative to gp40 and p24 FIV antigens) and for FeLV p27 antigen with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Snap FeLV/FIV Combo Plus Test; Idexx Laboratories, Hoofddorp, the Netherlands). Confirmatory tests were not performed as part of this study. Toxoplasma gondii sera IgG antibodies were detected using indirect fluorescent antibody tests (IFAT) performed with a commercial kit (Fuller-laboratories, Fullerton, CA USA). Titres ≥1:64 were considered seroreactive and, therefore, indicative of T gondii exposure.¹⁷

Statistical analysis

Fisher’s exact test and χ² analysis were used to test for associations between seropositivity for FIV, FeLV and T gondii and the animal source (urban/rural), gender (male/female), age (juvenile/adult), health status (healthy/unhealthy, presence of clinical abnormalities), CBC abnormalities and co-infection. Any parameters statistically linked to the ELISA seropositivity for FIV and FeLV, or to the IFAT seropositivity for T gondii IgG, were used in a logistic regression model to test for risk factors associated with the positive ELISA and IFAT test. Analyses were performed using a statistical software package (MedCalc version 9, Mariakerke, Belgium), with significance set at P <0.05.

Results

Sample characterisation

All 316 cats were tested for FIV and FeLV infection, and 203 cats were also tested for T gondii exposure. Results of the sample populations are shown in Table 1.

Table 1

Study population characteristics of 316 stray cats from colonies in northern Italy tested for FIV, FeLV infection and Toxoplasma gondii exposure during a trap-neuter-release (TNR) programme

Factor	Category	Number (%)
Origin of the cats (n = 316)	Urban area	226 (71.5)
	Rural area	90 (28.5)
Age (n = 315)	Juvenile	113 (35.9)
	Adult	202 (64.1)
Gender (n = 315)	Male	94 (29.8)
	Female	221 (70.2)
Health status (n = 314)	Healthy	129 (41.1)
	Unhealthy	185 (58.9)
Clinical abnormalities in unhealthy cats (n = 185)	Lymph node enlargement	130 (70.3)
	Pale mucous membranes	19 (10.3)
	Stomatitis	87 (47.0)
	Signs of respiratory tract infection	30 (16.2)
	Signs of ocular infection	45 (24.3)
CBC abnormalities (n = 297)	Anaemia	211 (71.0)
	Decreased Hb	48 (16.2)
	Decreased RBC	116 (39.1)
	Decreased PCV	193 (65.0)
	Leukopenia	38 (12.8)
	Leukocytosis	11 (3.7)
	Neutrophilia	20 (6.7)
	Neutropenia	26 (8.8)
	Lymphocytosis	6 (2.0)
	Lymphopenia	137 (46.1)
FIV test results (n = 316)	Positive	21 (6.6)
	Negative	295 (93.4)
FeLV test results (n = 316)	Positive	12 (3.8)
	Negative	304 (96.2)
T gondii test results (n = 203)	Positive	62 (30.5)
	Negative	141 (69.5)

CBC = complete blood count, FeLV = feline leukaemia virus, FIV = feline immunodeficiency virus, Hb = haemoglobin, PCV = packed cell volume, RBC = red blood cell

FIV seropositive cats

The overall FIV seropositivity was 21/316 cats (6.6%). FIV distribution by provenience, age, gender, health status, clinical and CBC abnormalities, and FeLV and T gondii status is presented in Table 2. Factors significantly associated with FIV seropositivity status included age, gender, decreased RBC count and co-infection with FeLV. Logistic regression analysis confirmed significant associations between FIV seropositivity and adulthood [odds ratio (OR) 5.9, 95% confidence interval (CI) 1.2848–27.3637, P = 0.0225], male gender (OR 5.4471, 95% CI 2.0312–14.6074, P = 0.0008) and decreased RBC count (OR 4.5393; 95%CI 1.6571–12.4347; P = 0.0033).

Table 2

Tests for a relationship between FIV-seropositive test results in stray cat colonies in northern Italy (21 cats) and risk variables using Fisher’s exact test

Factor	Category	FIV positive number (%)	P value of Fisher’s exact test	Odds ratio	95% CI
Origin of the cats	Urban area	16 (76.2%)	0.80	1.37	0.49–3.86
	Rural area	5 (23.8)	0.80	0.73	0.26–2.05
Age	Juvenile	2 (9.5)	0.01	0.17	0.04–0.76
	Adult	19 (90.5)	0.01 (0.02)*	5.76 (5.93)*	1.32–25.21
Gender	Male	13 (61.9%)	0.002 (0.0008)*	4.27 (5.45)*	1.71–10.69
	Female	8 (38.1)	0.002	0.23	0.09–0.59
Health status	Healthy	8 (38.1%)	0.82	0.87	0.35–2.18
	Unhealthy	13 (61.9)	0.82	1.14	0.46–2.84
Clinical abnormalities in unhealthy cats	Lymph node enlargement	10 (47.6%)	0.65	1.31	0.54–3.18
	Pale mucous membranes	2 (9.5)	0.37	1.71	0.37–7.95
	Stomatitis	3 (14.3%)	0.21	0.41	0.12–1.44
	Signs of respiratory tract infection	2 (9.5)	1.00	1.00	0.22–4.50
	Signs of ocular infection	5 (23.8)	0.20	1.98	0.69–5.69
CBC abnormalities	Anaemia	18 (85.7%)	0.14	2.58	0.74–9.00
	Decreased Hb	7 (33.3%)	0.06	2.87	1.09–7.53
	Decreased RBC	14 (66.7%)	0.01 (0.003)*	3.41 (4.54)*	1.33–8.73
	Decreased PCV	16 (76.2%)	0.35	1.79	0.64–5.03
	Leukopenia	1 (4.8)	0.49	0.32	0.04–2.48
	Leukocytosis	1 (4.8)	0.56	1.33	0.16–10.92
	Neutrophilia	3 (14.3%)	0.16	2.54	0.68–9.48
	Neutropenia	4 (19.0%)	0.10	2.72	0.84–8.78
	Lymphocytosis	0 (0.0)	1.00	0.97	0.05–17.76
	Lymphopenia	9 (42.9)	0.82	0.87	0.35–2.12
FeLV test results	Positive	3 (14.3%)	0.04 (0.07)*	5.30 (4.30)*	1.32–21.28
	Negative	18 (85.7)	0.04 (0.07)*	0.19	0.05–0.76
T gondii test results	Positive	5 (38.5%)	0.54	1.46	0.46–4.65
	Negative	8 (61.5)	0.54	0.69	0.22–2.19

P-values in bold are statistically significant (P <0.05)

Data from logistic regression analysis

CBC = complete blood count, CI = confidence interval, FeLV = feline leukaemia virus, FIV = feline immunodeficiency virus, Hb = haemoglobin, PCV = packed cell volume, RBC = red blood cell

Mean levels of haematological findings in FIV positive and negative cats are listed in Table 5 below.

FeLV seropositive cats

The overall FeLV seropositivity was 12/316 cats (3.8%). FeLV distribution by provenience, age, gender, health status, clinical and CBC abnormalities, and co-infection with FIV and T gondii are presented in Table 3. No correlations were detected between FeLV status and gender, age, origin, health status and haematological abnormalities. Co-infection with FIV was the only factor that showed a significant correlation with FeLV positivity (P = 0.04) based on Fisher’s exact test, but not with logistic regression analysis (OR 4.3046, 95% CI = 0.8470–21.8771, P = 0.0784).

Table 3

Tests for a relationship between FeLV-seropositive test results in stray cat colonies of northern Italy (12 cats) and risk variables using Fisher’s exact test

Factor	Category	FeLV positive (%)	P value of Fisher’s exact test	Odds ratio	95% CI
Origin of the cats	Urban area	8 (66.7%)	0.75	0.79	0.23–2.69
	Rural area	4 (33.3)	0.75	1.27	0.37–4.32
Age	Juvenile	3 (25.0%)	0.55	0.58	0.16–2.21
	Adult	9 (75.0)	0.55	1.71	0.45–6.45
Gender	Male	4 (33.3%)	0.76	1.18	0.35–4.03
	Female	8 (66.7)	0.76	0.85	0.25–2.88
Health status	Healthy	3 (25.0%)	0.37	0.47	0.12–1.75
	Unhealthy	9 (75.0)	0.37	2.15	0.57–8.09
Clinical abnormalities in unhealthy cats	Lymph node enlargement	6 (50.0%)	0.56	1.44	0.45–4.55
	Pale mucous membranes	1 (8.3)	0.53	1.43	0.18–11.73
	Stomatitis	6 (50.0%)	0.10	2.73	0.86–8.70
	Signs of respiratory tract infection	1 (8.3)	1.00	0.86	0.11–6.87
	Signs of ocular infection	3 (25.0)	0.39	2.06	0.54–7.93
CBC abnormalities	Anaemia	11 (91.7%)	0.19	4.67	0.59–36.78
	Decreased Hb	2 (16.7%)	1.00	1.04	0.22–4.90
	Decreased RBC	5 (41.7%)	1.00	1.12	0.35–3.62
	Decreased PCV	11 (91.7%)	0.06	6.23	0.79–48.91
	Leukopenia	1 (8.3)	1.00	0.61	0.08–4.86
	Leukocytosis	0 (0)	1.00	0.95	0.05–17.14
	Neutrophilia	0 (0.0)	1.00	0.52	0.03–9.07
	Neutropenia	2 (16.7%)	0.28	2.18	0.45–10.50
	Lymphocytosis	0 (0)	1.00	1.72	0.09–32.27
	Lymphopenia	7 (58.3)	0.56	1.67	0.52–5.38
FIV test results	Positive	3 (25.0%)	0.04 (0.07)*	5.30 (4.30)*	1.32–21.28
	Negative	9 (75.0)	0.04 (0.07)*	0.19	0.05–0.76
T gondii test results	Positive	3 (37.5%)	0.70	1.38	0.32–5.98
	Negative	5 (62.5)	0.70	0.72	0.17–3.12

Data from logistic regression analysis

CBC = complete blood count, CI = confidence interval, FeLV = feline leukaemia virus, FIV = feline immunodeficiency virus, Hb = haemoglobin, PCV = packed cell volume, RBC = red blood cell

Mean levels of haematological findings in FeLV positive and negative cats are listed in Table 5.

Toxoplasma gondii IgG seropositive cats

The overall prevalence of T gondii IgG antibodies was 62/203 cats (30.5%). Toxoplasma gondii seropositivity distribution by provenience, age, gender, health status, clinical and CBC abnormalities, and FIV and FeLV status are presented in Table 4. A χ² test did not detect any correlation between T gondii status and gender, age, origin, health status or haematological abnormalities.

Table 4

Tests for a relationship between Toxoplasma gondii IgG seropositivity in stray cat colonies from northern Italy (62 cats) and risk variables using a χ² test

Factor	Category	Number of T gondii positive (%)	P value of χ² test	Odds ratio	95% CI
Origin of the cats	Urban area	31 (50.0)	0.36	0.72	0.39–1.31
	Rural area	31 (50.0)	0.36	1.39	0.76–2.53
Age	Juvenile	23 (37.1)	0.42	0.74	0.40–1.37
	Adult	39 (62.9)	0.42	1.35	0.73–2.49
Gender	Male	13 (21.0)	0.34	0.66	0.33–1.35
	Female	49 (79.0)	0.34	1.51	0.74–3.08
Health status	Healthy	24 (38.7)	0.83	0.89	0.48–1.64
	Unhealthy	37 (59.7)	0.83	1.12	0.61–2.07
Clinical abnormalities	Lymph node enlargement	27 (43.6)	0.95	1.03	0.56–1.88
	Pale mucous membranes	2 (3.2)	0.77	1.15	0.21–6.47
	Stomatitis	16 (25.8)	0.92	1.03	0.52–2.04
	Signs of respiratory tract infection	8 (12.9)	0.20	2.20	0.80–6.00
	Signs of ocular infection	9 (14.5)	0.90	1.04	0.44–2.43
CBC abnormalities	Anaemia	50 (80.7)	0.10	2.00	0.95–4.24
	Decreased Hb	11 (17.7)	0.63	1.33	0.59–3.00
	Decreased RBC	23 (37.1)	0.61	1.24	0.66–2.33
	Decreased PCV	50 (80.7)	0.08	2.08	0.98–4.38
	Leukopenia	4 (6.5)	0.37	0.52	0.17–1.62
	Leukocytosis	2 (3.2)	0.95	1.48	0.24–9.09
	Neutrophilia	7 (11.5)	0.20	2.35	0.79–7.03
	Neutropenia	0 (0)	0.09	0.11	0.01–1.88
	Lymphocytosis	2 (3.3)	0.18	11.30	0.53–239.07
	Lymphopenia	27 (44.3)	0.94	1.07	0.58–1.98
FIV test results	Positive	5 (8.1)	0.74	1.46	0.46–4.65
	Negative	57 (91.9)	0.74	0.69	0.22–2.19
FeLV test results	Positive	3 (4.8)	0.96	1.38	0.32–5.98
	Negative	59 (95.2)	0.96	0.72	0.17–3.12

CBC = complete blood count, CI = confidence interval, FeLV = feline leukaemia virus, FIV = feline immunodeficiency virus, Hb = haemoglobin, PCV = packed cell volume, RBC = red blood cell

Mean levels of haematological findings in T gondii- positive and negative cats are presented in Table 5.

Table 5

Haematological findings (mean levels ± standard deviation SD) in feline immunodeficiency virus (FIV), feline leukaemia virus (FeLV) and IgG Toxoplasma gondii seropositive and seronegative cats in stray cat colonies from northern Italy

Parameter	Reference interval	Unit	Mean patient data ± standard deviation (SD)
			FIV positive		FIV negative		FeLV positive		FeLV negative		IgG T gondii positive		IgG T gondii negative
			Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
RBC	6000–10100	× 10³/µl	5734	1527	6444	1197	6097	1630	6406	1217	6398	1289	6574	1211
Hb	8.1–14.2	g/dl	8.63	1.81	9.51	1.61	9.04	1.99	9.47	1.63	9.41	1.76	9.69	1.67
PCV	27.7–46.8	%	24.05	5.24	26.29	5.27	24.47	4.86	26.20	5.30	24.45	4.69	25.56	4.47
WBC	6300–19600	/µl	10563	4371	10694	4523	9354	3809	10742	4529	11439	4847	10605	3743
Neutrophil	3000–13400	/µl	7074.2	3790.4	7241.5	3928.5	6714.5	2933.8	7251.4	3951.3	8402.6	4046.2	7976.6	3327.0
Lymphocyte	2000–72000	/µl	2398.3	1495.2	2500.2	1691.4	1759.9	845.0	2523.9	1676.9	2443.7	1696.0	2038.3	1200.7

Hb = haemoglobin, PCV = packed cell volume, RBC = red blood cell count, SD = standard deviation, WBC = white blood cell

Co-infection

Three of 316 samples (1%) were positive for both FIV and FeLV. Out of 203 samples tested for FeLV, FIV and T gondii, five (2.5%) were positive for FIV and T gondii, and three (1.5%) were positive for FeLV and T gondii. No samples were positive to all three agents. The statistical association between FIV and FeLV detected by the Fisher’s exact test was not confirmed by the logistic regression analysis. No statistical association was observed between FIV or FeLV occurrence and T gondii co-infection.

Discussion

This study investigated the seroprevalence of FIV and FeLV infection, and T gondii exposure in stray cat colonies of northern Italy. In Italy, National Law number 281 of 14 August 1991 on the management of pets and on the control of stray cats introduced a no-kill policy for this species.¹⁸ Thus, TNR programmes have been carried out to control stray cat populations in many cities of Italy. TNR programmes are considered the most practical, effective and humane way of controlling free-roaming cats because they are intended to decrease reproduction without causing harm to the cats.¹⁹ In Milan, there are more than 450 feline colonies.²⁰ It is common for people to care for these stray cats without assuming full ownership of them. This scenario allows for daily direct contact with these cats during feeding, as well as indirect contact through fomites, such as clothes and shoes, and with their pet cats. People may also contact stray cat colonies at schoolyards and public gardens. Stray cats from colonies are susceptible to infections by viruses of different families, and viruses belonging to the family Retroviridae often cause persistent infections. Controlling transmission of these viruses is difficult because they are transmitted directly, causing both chronic and subclinical infections.²¹ Knowledge of FIV and FeLV prevalence in stray cat colonies is important to assess the welfare of the cats themselves, and to assess the threat they pose to pet cats with outside access.

The overall prevalence of FIV infection in our population was 6.6%. Adult age, male gender and decreased RBC count were significant predictors of FIV seropositivity. The prevalence of FIV infection among adult males was compatible with the knowledge that fighting and biting behaviour of stray cats in colonies is the primary mode of transmission of this infection. FIV is effectively transmitted via bite wounds and higher cat densities would lead to more inter-cat aggression and fighting. Male cats were predisposed to FIV infection as transmission of FIV is mainly through bite wounds, and male cats, irrespective of their neutering status, are more likely to show territorial aggression and be involved in fights. It is interesting to note that FIV-positive status in our study was significantly associated with reduced RBC count, even though other red cell indices (PCV and Hb) were not associated. This result could be a consequence of the small number of cats sampled.

No association was evident between FIV infection and health status. FIV can cause an acquired immunodeficiency syndrome that increases the risk of developing opportunist infections, neurological diseases and tumours.²² A possible justification for the lack of an association between FIV infection and health status is that FIV in most naturally infected cats does not cause severe clinical signs and FIV infected cats may live many years without any health problems. In addition, it is possible that some clinical signs, such as those related to neurological signs or tumours, would not be evident in the cats of our study owing to a lack of historical reference data and because the cats were evaluated under general anaesthesia.

The worldwide data on FIV and FeLV prevalence showed a great variation within and between countries. This variability could be caused by a number of factors, including differences in population densities, the level of neutering, age, gender ratios and interaction between cat colonies. The overall prevalence of FIV in our study was lower than other studies conducted in Italy on stray cat colonies; these estimates ranged from 7% of 490 cats in the Veneto region to 35% of 99 cats from northern Italy.^6,7 The only epidemiological data for Milan city estimated a seroprevalence of 13.3%, but only 30 stray colony cats were tested.²³ Our FIV prevalence was, however, similar to seroprevalence in stray cats in Europe ranging from 6.6% of 196 free roaming cats in Finland to 11.3% of 346 urban stray cats in Belgium.^5,8 Finally, our prevalence estimates were higher than the 5.2% of 533 stray cats in north Florida and 2.4% of 585 stray and feral cats in Maryland (USA).^4,10

It has been recommended that positive results for the FIV ELISA be confirmed with a Western blot assay.²¹ Reported sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the assay used in our study to detect FIV antibodies was 100%, 99.6%, 94.5% and 100% respectively.²⁴ Given this information, if a false-positive occurred in our study it should be limited, as the specificity and PPV of the assay used were nearly 100%. As the ELISA used in this study for assessment of the antibodies to FIV had a sensitivity of 100%, no false-negative results to FIV infection should be expected.

Nevertheless, factors that might lead to a bias could include seropositivity to FIV from maternally-derived FIV antibodies and seropositivity following vaccination against FIV infection. The possible presence of maternally-derived antibodies to FIV in kittens younger than 6 months of age are reported to confound interpretation of positive test results.²⁵ Prevalence of FIV positivity in young cats in our study was low (2/21 cats) and a statistically significant value was found for adult age when compared with positivity in juvenile cats. Therefore, it seems likely that bias of FIV prevalence estimates caused by the possible presence of maternal antibodies was minimal. In addition, administration of the FIV vaccine can result in positive test results through production of antibodies that induce positive ELISA results.²⁶ The vaccine against FIV has not been licensed and introduced in Italy so no bias for vaccination should be present in our population. In our study, another bias could have affected real seroprevalence for FIV if cats had not yet seroconverted following recent exposure and when FIV-specific antibodies in serum are less than the detection limit of the test.

In contrast to FIV infection, in which prevalence varies significantly, the FeLV infection rate of free-roaming cats is similar throughout the world, ranging from 1% to 8%, and our results support these data. The overall prevalence of FeLV in our study was slightly higher than the 2.3% of 136 stray cats in Piedmont, Liguria and Val d’Aosta.^3,27 In the only study that tested 30 colony cats from Milan, none were detected as positive for FeLV.²⁸ In Europe, prevalence of FeLV in stray cats ranged from 1% of 196 free roaming cats in Finland to 7.1% of 198 stray cats in Lisbon.^5,12 In the USA, FeLV seropositivity was detected in 4.3% of 1876 free roaming cats from North Carolina and Florida.⁹ In our study, cats with retrovirus infection came predominantly from urban areas. Although it was not statistically significant, such a result may arise because urban animals lead more colonial lifestyles, whereas cats living in rural areas probably do not have as much intraspecific contact, thereby limiting opportunities for horizontal disease transmission characteristic of retroviruses infections. FeLV is more pathogenic than FIV, and was long considered to be responsible for more clinical syndromes (mainly anaemia) and subsequent secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system.²² The lack of association between health status and FeLV infection in our survey could be related to the small number of cats that were FeLV-positive and, as for FIV, owing to a lack of data about health history and because the cats were evaluated under general anaesthesia.

Reported values for sensitivity, specificity, PPV and NPV of the FeLV test were 92.3%, 97.3%, 73.5% and 99.4% respectively.²⁴ Positive results for the FeLV ELISA should be confirmed with an immunofluorescence antibody test.²⁹ Because positive assay results were not confirmed by means of an alternative assay in the present study, it is possible that some positive assay results were falsely positive for infection. False-negative results were also possible because the sensitivity of the test used was not 100%. Negative assay results are also possible and occur when the concentration of FeLV p27 antigen in serum is less than the detection limit of the test or during the pre-antigenaemic stage of infection. A bias that could have lowered the real prevalence of natural FeLV infection in our population is the possibility that colony cats had received prior vaccination against FeLV. In addition to costs associated with vaccinating feral cats, other considerations on the utility of vaccination of colony cats include uncertainty about what proportion of free-roaming cats are naïve to infectious diseases and would benefit from vaccination; whether administration of a single vaccine is beneficial; and whether cats can mount an adequate immune response when they are stressed by capture, transportation, anaesthesia and surgery.³⁰ For these reasons, FeLV vaccine, even if available in Italy, was not used routinely in stray colony cats in Lombardy, so no bias regarding the vaccination against FeLV infection could have affected our population. Finally, our FeLV infection prevalence results could potentially be biased if vaccinated domestic cats became integrated into the feral population.

The overall prevalence for T gondii IgG antibodies in our study was 30.5%. This seroprevalence was in agreement with those reported in stray populations of cats from other authors in Italy with prevalence higher than 30%.^7,11,31 Compared with seroprevalence in stray cats in Europe, our data is intermediate between the lowest prevalence of 24.2% found in Lisbon and the highest (70.2%) found in Belgium.^8,12 Our prevalence is similar to 31.6% of sick domestic cats in a large feline population in USA.³² Although previous studies have recognised an association between infections with FIV and T gondii exposure, no association was observed in this study as nearly all T gondii IgG positive cats were negative for FIV and FeLV infection.⁴ In our study, concurrent retroviral infections were not found to be associated with increased risk for developing T gondii antibodies. As seropositive cats have likely already shed T gondii oocysts, and these oocysts can remain viable in the environment for many months, serological surveys of anti- T gondii antibodies in these animals could be helpful in identifying the proportion of cats that have a role in environmental contamination with faecal oocysts.¹⁷ The high prevalence of toxoplasmosis exposure in a large stray cat population may seriously contaminate the urban environment. The accidental ingestion of oocysts by contact with soil was found to be among the main factors associated with the infection of pregnant women in European cities, with between 6% and 17% of infections in different cities attributed to soil contact; women from Milan city also participated in this study.³³

In conclusion, information gathered from FeLV and FIV testing in stray colony cats in northern Italy showed that colony cats do not appear to pose a great risk for retrovirus infection to pet cats allowed outdoors. This low prevalence could be owing to a history of management of this population through neutering procedures that preserve the social structure of the cat population and decrease competition for food, shelter and territory. On the contrary, toxoplasmosis prevalence rates in our study suggested that exposure to T gondii in stray cats is common and these cats may play an important role in the contamination of the environment with the oocysts. Informing the public about the risk of infection through contact with soil and reducing the populations of stray cats by spaying in TNR programmes may represent measures to reduce the risk of human infection. Also, provision of commercial food for stray cat colonies, as practised by the Milan Municipality for a number of years, could help limit the spread of T gondii into the environment through discouragement of hunting.

Lastly, prevalence rates for both retroviral and T gondii infections may change over time and, therefore, similar surveys should be repeated to monitor trends. Additional studies and up-to-date data are therefore required for a more complete understanding of feline infections in Europe.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors do not have any potential conflicts of interest to declare.

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