15

Parole chiave
Gestione degli alveari,
Inquinamento 
ambientale,
Mortalità delle api,
Monitoraggio,
Aree Naturali Protette.

Riassunto
Il presente studio riporta i risultati del monitoraggio dello stato di salute delle api 
effettuato da ottobre 2009 a dicembre 2010 in cinque Aree Naturali Protette italiane, 
scelte per rappresentare le aree biogeografiche alpina, continentale e mediterranea. 
All'interno di ciascuna è stato posizionato un apiario di 20 alveari vicino a potenziali fonti 
di inquinamento (ad esempio aree agricole, aree industriali o insediamenti urbani) e un 
altro apiario di 20  alveari lontano da possibili fonti di inquinamento. Per monitorare lo 
stato di salute delle api, è stata messa in relazione la mortalità degli alveari con la presenza 
di malattie delle api, con l'ambiente (Naturality Index, presenza di prodotti fitosanitari ed 
esposizione a metalli pesanti) e la gestione dell’apiario. Non sono stati osservati effetti 
significativi degli inquinanti di origine antropica e dei patogeni sulla mortalità degli alveari, 
mentre la capacità di gestione degli alveari da parte degli apicoltori è risultata strettamente 
correlata alla mortalità delle colonie.

Monitoraggio dello stato di salute delle api
in cinque Aree Naturali Protette italiane

Keywords
Apiary management,
Environmental pollution,
Honey bee mortality,
Monitoring,
Natural protected area.

Summary
The health status of the honey bee populations has attracted a great amount of interest 
in recent years. We investigated honey bee health in five natural protected areas in Italy 
from October 2009 to December 2010. Areas were selected to represent a wide range of 
bio‑geographical zones including alpine, continental, and Mediterranean. Within each of 
these natural protected areas, one apiary of 20 colonies near potential pollution sources (e.g., 
agricultural areas, industrial areas, or urban settlements) and another apiary of 20  colonies 
far from possible sources of pollutants have been placed. To monitor honey bee health, 
colony mortality was related to: honey bee pathologies, environment (Naturality Index, plant 
protection products and heavy metal exposure), and apiary management. Anthropogenic 
pollutants and pathogens did not have significant effects on colony mortality while 
environment and the poor colony management skills of the beekeepers did.

Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4
Accepted: 11.09.2017  |  Available on line: 06.03.2019

1Istituto Superiore per la Protezione e la Ricerca Ambientale, Via Vitaliano Brancati 60, 00144 Rome, Italy.
2Istituto Zooprofilattico Sperimentale del Lazio e della Toscana ‘M. Aleandri’,

Via Appia Nuova 1411, 00178 Rome, Italy.
3Dipartimento di Scienze Veterinarie, Università degli Studi di Pisa, Viale delle Piagge 2, 56124 Pisa, Italy.

4Dipartimento di Scienze e Tecnologie Agro-Alimentari (DISTAL), Alma Mater Studiorum, Università di Bologna,
Via Zamboni 33, 40126 Bologna, Italy.

5Istituto Zooprofilattico Sperimentale delle Venezie, National Reference Laboratory for Beekeeping,
Viale Dell’Università 10, 35020 Legnaro (PD), Italy.

*Corresponding author at: Istituto Zooprofilattico Sperimentale del Lazio e della Toscana ’M. Aleandri’, Via Appia Nuova 1411, 00178 Rome, Italy.
Tel.: +39 06 79099328, e-mail: giovanni.formato@izslt.it.

Valter Bellucci1, Stefano Lucci1, Pietro Bianco1, Alessandro Ubaldi2, Antonio Felicioli3,
Claudio Porrini4, Franco Mutinelli5, Sabrina Battisti2, Valentina Spallucci2, Antonella Cersini2,

Marco Pietropaoli2 and Giovanni Formato2*

Monitoring honey bee health
in five natural protected areas in Italy



16 Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Honey bee health in Italy Bellucci et al.

Materials and methods
The monitoring project was conducted from October 
2009 to December 2010 using 200 honey bee colonies 
within five Italian NPAs (Figure 1) as representative of 
a wide range of the bio‑geographical regions in Italy 
including alpine, continental, and Mediterranean 
areas. These NPAs were all located near agricultural, 
industrial, or urban settlements and are listed as 
follows:

• Parco Nazionale delle Dolomiti Bellunesi 
(Dolomiti), alpine bioregion, Northeastern 
Italy;

• Parco dei Gessi bolognesi e dei Calanchi 
dell’Abbadessa (Calanchi), continental bioregion, 
Northern Italy;

• Parco di Migliarino San Rossore Massaciuccoli 
(San Rossore), Mediterranean bioregion, Central 
Italy;

• Parco dei Monti Simbruini (Simbruini), 
Mediterranean bioregion influenced by 
sub‑continental conditions, Central Italy;

• Riserva Naturale Statale Litorale Romano 
(Litorale), Mediterranean bioregion, Central Italy.

For the present study, two apiaries of 20 healthy 
colonies were established in each NPA (40 colonies/
NPA). One apiary was classified as a ‘non‑exposed’ 
and called ‘Apiary A’ (combined with site names 
as Dolomiti A, Calanchi A, San Rossore A, Litorale 
A, Simbruini A) and located in ecosystems with 
a modest level of anthropogenic pressure. The 
other apiary was classified as ‘exposed’ and called 

Introduction
The decline of honey bee (Apis mellifera L.) colonies, 
observed in the last few decades in both Europe 
and in the United States (Ellis et al. 2010, Potts et al. 
2010), have a multi‑factorial origin (Neumann and 
Carreck 2010) including Plant Protection Products 
(PPPs), beekeeping practices, pest and pathogens, 
queen failure, genetic weakness, nutrition, and 
weather patterns.

Farming techniques and crop protection procedures 
play a pivotal role in the proper management of 
honey bee colonies and their possible exposure 
to PPPs (Alaux et  al. 2010, Brodschneider and 
Crailsheim 2010, Johnson et al. 2010). Environment 
pollution, especially pesticides, can negatively 
affect the health of honey bee colonies. Numerous 
studies have reported on the negative effects of 
exposure to PPPs on honey bees (Koch and Wisser 
2001, Forster 2009, Anne and Gavin 2010, EFSA 
2012, Henry et  al. 2012, Whitehorn et  al. 2012). In 
addition, honey bee pests and pathogens probably 
play a crucial role in honey bee colony losses, 
especially losses caused by Varroa destructor 
(V.  destructor) combined with viruses and Nosema 
ceranae (vanEngelsdorp et  al. 2009, Neumann and 
Carreck 2010, Martin et  al. 2013). The interaction 
of these pathogens with pesticides also causes 
losses of hives (Pettis et al. 2012, Pettis et al. 2013). 
Aside from PPPs, honey bee pests and pathogens, 
even beekeeping management contributes to 
endangering the status of honey bee health 
(Oldroyd 2007, vanEngelsdorp et al. 2008).

About 75,000 beekeepers in Italy manage more 
than 1,317,000 colonies [Commission Implementing 
Regulation (EU) N. 768/2013]. The responses 
from 874 surveys conducted in 2007‑2009 by the 
organization named Prevention of honeybee COlony 
LOSSes, using the COST Action FA0803 framework, 
showed Winter mortality ranged from 11% (Abruzzo) 
to 38% (Emilia Romagna), with an average of 23.5% 
(Mutinelli et  al. 2010, van der Zee et  al. 2012). A 
pan‑European epidemiological study on honeybee 
colony losses called EPILOBEE (Laurent et  al. 2015), 
set up from 2012 to 2014 in 17  European Member 
States, showed a overwintering colony mortality in 
Italy ranging from 5.5% (winter 2012‑2013) and 4.8% 
(winter 2013‑2014).

This study, coordinated by the Italian National 
Institute for Environmental Protection and Research 
was promoted in 2009‑2010 by the Italian Ministry 
of Environment, Territory and Sea to evaluate 
honey bee mortality within five natural protected 
areas (NPAs) in Italy, in order to verify the effects of 
chemical pollution, apiary management, and bee 
pathogens on honey bee’s health. Figure 1. Locations of the five natural protected areas included in the 

survey.

Dolomiti Bellunesi

Simbruini

Calanchi Bolognesi

Litorale Romano

San Rossore



17Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Bellucci et al.  Honey bee health in Italy

used 1 week as a unit of time; this corresponds to 
the weekly beekeeper check of hive status. The 
differences between the survival curves of A and B 
colonies of the five apiaries were evaluated through 
a log‑rank test (Thrusfield 1995). The association of 
both the cumulative and winter mortality indices 
with honey bee diseases and the weekly count of 
the under‑basket mortality (>  200 bees/colony) 
was measured by the Spearman’s rank correlation 
coefficient (r

s
); p  <  0.05 was selected as the level 

of significance. STATA 12.0 software was used for 
statistical analysis. 

The honey bee diseases investigated were: 
Varroasis, seven main honey bee viruses (Acute Bee 
Paralysis Virus, ABPV; Black Queen Cell Virus, BQCV; 
Chronic Bee Paralysis Virus, CPBV; Deformed Wings 

‘Apiary B’ (Dolomiti B, Calanchi B, San Rossore B, 
Litorale B, Simbruini B)and located close to potential 
anthropogenic pollutant sources with agricultural, 
industrial, and/or urban contaminants. Apiaries A 
and B were established in similar climatic conditions.

Data sheets were prepared to standardize data 
collection related to colony inspections (health 
status and strength), honey bee mortality, samplings, 
and the environment. Supplementary inspections 
and samplings of honey bees and pollen were 
performed in case of variations in beehive health 
status (e.g., colony depopulation, disease, death, 
higher mortality of adult honey bees) detected by 
the beekeepers.

Laboratory analyses were carried out in an accredited 
laboratory in conformity with UNI CEI EN ISO/IEC 
17025 (International Organization for Standardization 
and the International Electrotechnical Commission 
international standards). To assess the health status 
and the strength of the colonies for each apiary, 
four ad hoc trained inspectors conducted quarterly 
clinical inspections. Moreover, bee mortality was 
assessed weekly.

Colony mortality was measured as ‘cumulative 
mortality’, ‘winter mortality’, and ‘mortality rate’. A 
dead colony was assessed if no honey bees of the 
colony were found alive. ‘Cumulative mortality’ 
indicates the ratio of the number of dead colonies 
in each apiary found throughout the entire 
observational period (1 year) and the number 
of colonies (20) monitored in each apiary at the 
beginning of the project. ‘Winter mortality’ indicates 
the ratio of the number of dead colonies in each 
apiary found throughout the winter season (from 
1 October to 1 April) and the number of colonies 
(20) monitored in each apiary at the beginning 
of the project (1 October). The ‘mortality rate’ 
(colony‑month at risk) indicates the mortality rate 
calculated on a monthly basis during the entire 
follow‑up period (Thrusfield 1995).

Under‑basket cages were used as a supplementary 
tool to monitor bee mortality within each colony 
(Human et al. 2013). These cages were placed in front 
of each colony for a weekly count of the number of 
dead adult honey bees in each colony. Whenever the 
number of dead honey bees exceeded the threshold 
of 200 honey bees/week in the same colony (Porrini 
et  al. 2003), an additional inspection of the colony 
was combined with samplings for pathogens and 
pollutants (PPPs and heavy metals) to find the 
cause of the increased mortality. To compare data 
related to cumulative mortality, survival curves in 
exposed and non‑exposed colonies were drawn 
using the non‑parametric method of Kaplan‑Meier 
(Kaplan and Meier 1958). This model represents the 
survival function as the probability that a colony 
will survive over a given period. The present study 

Table I. Pathogens, contaminants, methods and matrices used to 
monitor the honey bee health of the apiaries located in the natural 
protected areas.

Pathogen/
contaminant Diagnostic methods Matrix

Varroasis
(Varroa destructor)

Visual identification of 
the parasite

Adult honey bees with 
symptoms of disease;

honey bee brood of 
hives with symptoms 

of disease
Main honey bee 

viruses:
DWV, ABPV, CPBV, 

BQCV, SBV, KBV, IAPV

Reverse Transcriptase 
- Polymerase Chain 
Reaction (RT-PCR)
(Singh et al. 2010)

Adult honey bees
(10 adult honey bees)

American Foulbrood 
- AFB

(Paenibacillus larvae)

Cultural method
(OIE 2008)

Honey bee brood
(3-5 affected larvae)

European Foulbrood 
- EFB

(Melissococcus 
plutonius)

Cultural method
(OIE 2008)

Honey bee brood 
with symptoms of the 

disease

Nosemosis
(N. apis and 
N. ceranae)

Polymerase Chain 
Reaction (PCR) 

and microscopic 
examination

(OIE 2008)

Adult honey bees
(10 adult honey bees 
and 30 honey bees, 

respectively)

Ascosphaera apis Cultural method
Honey bee brood
(affected larvae)

Main Plant Protection 
Products (PPPs): 
Organochlorine, 

Organophosphorous, 
Pyrethroids, 

Neonicotinoids and 
Carbamates

High resolution gas 
chromatography 

separation analyses 
method for 

neonicotinoids

Adult honey bees
(200 bees/hive)

High resolution gas 
chromatography

Honey
(500 g/apiary)

High resolution gas 
chromatography

(for neonicotinoids 
only)

Pollen
(10 cc)

Heavy metals: Pb, Cd, 
Cr and Cu

Atomic absorption 
spectrophotometry

Honey
(500 g/apiary)

Palynological analysis Optical microscopy
Pollen

(10 g/colony)



18 Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Honey bee health in Italy Bellucci et al.

adopted for analyzing PPPs. Because neonicotinoid 
compounds are thermally unstable, high‑resolution 
gas chromatography separation analysis was 
preferred instead of high‑resolution liquid 
chromatography. Palynological analyses were used 
to identify pollen grains using optical microscopy 
(Model B‑500tph, Optika Srl, Ponteranica, BG, Italy) 
on the beebread samples from dead or depopulated 
colonies, so as to relate the poisonings of honey 
bees by PPPs to the treated plants.

To evaluate the apiary management, a score from 
1 (weak) to 5 (excellent) was given to the beekeeper 
skills, considering both their commitment to 
beekeeping and their ability to adopt good 
beekeeping management practices. The grades 
were assigned according to an evaluation grid 
designed and approved by the working group 
(Table II). In four NPAs, the two apiaries were 
managed by different beekeepers according to their 
customary practices. For the San Rossore NPA only, 
the same beekeeper managed both apiaries. All the 
beekeepers had to follow the same measurement 
protocols. The relationship between the skill of 
beekeepers (beekeepers score) and the mortality 
indices (cumulative mortality, winter mortality) 
was measured by the Spearman’s rank correlation 
coefficient. Then, linear regression was used to 
measure their reciprocal influence.

Results
A total of 826 clinical inspections of beehives and 
733 laboratory analyses were conducted to check 
the health status of honey bees and their exposure 
to pollutants. Table III shows detailed data about 
the analytical activities conducted for the project. 
Table IV reports the results of hive mortality for the 
ten apiaries as cumulative mortality, mortality rate, 
and winter mortality.

The colonies of the B apiaries of Litorale and San 
Rossore Parks showed higher mortality compared 
with the corresponding A apiaries.

The log‑rank test applied on Kaplan‑Meier curves 
representing aggregated data of colony mortality 
rates according to apiary exposure to pollution 

Virus, DWV; Israeli Acute Paralysis Virus, IAPV; Kashmir 
Bee Virus, KBV; Sac Brood Virus, SBV), American 
Foulbrood (Paenibacillus larvae), Ascosphaera apis, 
European Foulbrood (Melissococcus plutonius), and 
nosemosis (Nosema apis and Nosema ceranae). 
Table I provides the laboratory methods adopted 
for the above‑mentioned analyses. To compare 
the frequency of each infectious and parasitic 
disease within exposed and non‑exposed apiaries, 
a series of 2  ×  2 tables for each disease and NPAs 
was developed and the Fisher’s exact test was 
conducted. When appropriate, risk measure was 
expressed as a risk ratio (RR). When the colonies 
experienced depopulation, death, relatively high 
mortality of adult bees or honey bee pathologies, 
extra‑inspections and extra‑samplings were 
conducted to assess chemical and/or biological 
causes of the related problems.

To investigate the relationships between honey bee 
mortality and environment, data were collected 
related to a Naturality Index, including land use, wild 
vegetation, and crops. Farming techniques were 
also recorded. Around each apiary, a 1.5‑km radius 
buffer area (honey bee flight area) was evaluated; 
maps of land use and vegetation coverage were 
produced using a scale of 1:10,000. Land use and 
vegetation polygons were delineated using both 
photo interpretation and field surveys. The identified 
polygons were referred to the European University 
Information Systems, Co‑ordination of Information 
on the Environment Biotopes, and Natura 2000 
(Council Directive 92/43/EEC) categories according 
to the European Environment Agency (EUNIS 2007).  
The current agricultural use (presence of vegetable 
crops, vineyards, and corn) was extrapolated in detail 
from these buffer maps. The number of vegetation 
categories and the surface area for each of them 
were calculated with the Naturality Index (expressed 
as natural + natural like/urban + agricultural 
surfaces), Shannon Diversity Index, and the Simpson 
Dominance Index. Naturality Index  was associated 
with cumulative mortality through Pearson chi 
square test with Yates correction, furthermore a 
linear regression was used to analyze correlation 
between cumulative and agricultural land coverage.

Honey samples were collected monthly from 
each colony to analyze any heavy metal residues 
of Pb, Cd, Cr, and Cu with atomic absorption 
spectrophotometry (Table I). To test for differences in 
the average concentration of heavy metals in honey 
taken from the A and B apiaries, the Student’s t‑test 
was performed for each NPA and each individual 
metal. To investigate the residues of PPPs, adult bees 
and honey were sampled monthly from each apiary. 
Moreover, the same types of samples were collected 
from each colony in all cases of abnormally high 
colony mortality to monitor the exposure to PPPs 
and heavy metals. Table I provides the methods 

Table II. Scoring criteria used to evaluate the management commitment 
and the success in adopting good beekeeping practices. Scores range 
from 1 (weak) to 5 (excellent).

Commitment
Excellent Sufficient Weak

Management
Excellent 5 4 2
Sufficient 4 3 2

Weak 2 2 1



19Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Bellucci et al.  Honey bee health in Italy

The comparison of the cumulative mortality 
between colonies placed in buffers at different 
Naturality Index values showed higher values for 
cumulative mortality in areas with a lower Naturality 
index (Figure 4). This difference resulted significant 
(p = 0.047)

Table VII shows the amounts of several heavy metals 
found in the honey samples; the statistical analysis 
showed no significant differences between exposed 
(area B) and non‑exposed (A) apiaries. In addition, no 
residues of PPPs were found in the monthly honey 
samples with the exception of one case where 
the active principle imidacloprid was detected in 
a dead honey bee sample, at a concentration of 
0.0096  mg/kg [equivalent to about ¼ of the Lethal 
Dose 50% (LD

50
) for honey bees], in March 2010, in 

the non‑exposed Apiary A of Calanchi.

Varroosis was detected at different levels in all the 
apiaries (Table VIII). The Spearman’s rank correlation 
coefficient used to measure the relationship 
between heavy Varroa infestation and the mortality 

(Figure 2) showed that the mortality rate detected 
in the exposed (B) apiaries was significant higher 
compared to the mortality rate detected in the 
non‑exposed (A) apiaries (p = 0.0002). Colony survival 
on the 30th week (210 days) was 87% for non‑exposed 
and 75% for exposed apiaries. Table  V reports the 
excesses of adult honey bee weekly mortality (> 200 
dead honey bee) found in the under‑basket cages, 
for each individual hive. This parameter was not 
observed to be related to colony mortality (both 
cumulative and winter mortality) in our study.

The vegetation surveys conducted in the honey bee 
flight area provided a critical contribution to the 
study. Table VI shows the synthetic characteristics of 
land use, vegetation coverage, naturalness, and the 
diversity of the above‑mentioned buffer zones. In 
all five NPAs, linear regression showed only a weak 
correlation (r

2
 = 0.0373) between increased mortality 

of the colonies with an increase in agricultural land 
coverage (cultivation of carrots, forage crops, mixed 
crops, horticultural crops, alfalfa, corn, melons, 
olive plantations, potatoes, rape, savoy cabbage, 
sorghum, vineyards, and watermelon; Figure 3) 
index of the possible use of PPP.

Table III. Activities performed to verify the health status of honey bees 
and their exposure to pollutants.

Activities Numbers
Clinical inspections of the hives 826

Samples for honey bee viroses 117

Samples for nosemosis 108

Samples for American Foul Brood (AFB) 24

Samples for European Foul Brood (EFB) 1

Samples for Ascosphaera apis 3

Samples for neonicotinoids 109

Samples for other PPPs 123

Samples for heavy metals 96

Palynological analyses 27

Table IV. Mortality rates (cumulative and winter mortalities) observed 
in non exposed and exposed apiaries located in natural protected areas.

Apiary Do
lo

m
iti

Ca
la

nc
hi

Li
to

ra
le

Sa
n 

Ro
ss

or
e

Si
m

br
ui

ni

A B A B A B A B A B
Cumulative 
mortality 15% 15% 0 0 20% 70% 25% 70% 5% 45%

Winter 
mortality 5% 15% 0 0 15% 45% 25% 70% 5% 45%

A = non exposed apiaries;    B = exposed apiaries.

0.00
0 100 200

Time (days)

Su
rv

iv
al

 p
ro

b
ab

ili
ty

 (%
)

Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct

300 400

0.25

0.50

0.75

1.00
Non exposed apiaries Exposed apiaries

Figure 2. Kaplan-Meier survival estimate curves for apiaries that were 
exposed and not exposed to high levels of pollutants.

Table V. Weekly mortality threshold excess found in the under basket 
cages of the apiaries located in selected natural protected areas (NPA).

NPA

Excesses of weekly 
mortality threshold
(> 200 honey bees/

hive/week)

Month

Simbruini A 2 May (1st week)

Simbruini B 0 -

Litorale A 9 May (1st and 2nd week)

Litorale B 10
November (2nd week)
December (4th week)

August (1st week)
Dolomiti A 0 -

Dolomiti B 2
May (4th week) 
June (4th week)

Calanchi A 0 -

Calanchi B 1 October (1st week)

San Rossore A 1 May (3rd week)

San Rossore B 1 October (3rd week)



20 Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Honey bee health in Italy Bellucci et al.

65 samples of honey bees (Table VIII). The frequency 
of the seven viruses did not significantly differ 
between A and B apiaries, with the exception of 
the following five cases: ABPV in Calanchi, CBPV in 
Dolomiti and Simbruini, KBV in Litorale, and SBV in 
Calanchi. However, the high variability of the RR 
did not allow any statement on a presumed major 
risk of viral diseases in the B apiary areas compared 
with the A ones. The Spearman’s rank correlation 
coefficients (r

s
) used to measure correlation among 

the prevalence of the seven main honey bee 
viruses listed above and the cumulative and winter 
mortality indices showed that the variables are 
unrelated for most of the cases with the exception 
of ABPV and KBV. Acute Bee Paralysis Virus was 
positively and significantly related with both 
cumulative and winter mortality (r

s
  =  0.6862 and 

0.6790, p  =  0.028 and 0.031, respectively). Kashmir 

indices (cumulative mortality, winter mortality) 
showed no correlation.

American Foulbrood (AFB) was detected in two 
colonies of the non‑exposed Dolomiti Apiary A, 
in three colonies of the Litorale Apiary A, in four 
colonies of the Litorale Apiary B, and in nine colonies 
of the Simbruini Apiary B (Table VIII). The prevalence 
of AFB ranged from 0 to 45% between the apiaries, 
with an average of 5% in the A apiaries and of 
13% in B apiaries. The Spearman’s rank correlation 
coefficient calculated between the frequency of 
AFB and both cumulative and winter mortality 
showed no correlation between them. However, 
no case of European Foulbrood (EFB) was detected 
during the study.

Seven main honey bee viruses (ABPV, BQCV, CBPV, 
DWV, IAPV, KBV, and SBV) were investigated in 

Table VI. Characteristics of the honey bee flight areas used for statistical analyses.

Dolomiti Calanchi Litorale San Rossore Simbruini
A B A B A B A B A B

N. Polygons 491 1,806 610 789 273 333 120 298 748 1,381

Average size of polygons (ha) 1.44 0.39 1.16 0.90 2.61 2.12 5.94 2.37 0.94 0.53

N. CORINE/EUNIS Categories 30 30 40 39 32 39 29 34 33 42

N. Natural categories 16 7 18 16 15 20 21 14 17 16

Shannon Diversity Index* 2.73 2.1 1.80 2.23 2.64 2.24 2.39 2.23 2.30 1.80

Simpson Dominance Index** 0.03 0.12 0.04 0.06 0.06 0.15 0.05 0.01 0.06 0.08
Rate naturalness
(Natural+ semi-natural/Agricultural+Urban)

6.32 0.74 1.38 0.29 0.53 0.98 8.68 0.34 6.79 1.04

% Forests 73.93 17.54 20.90 13.89 6.24 18.24 37.51 10.27 56.27 45.16

% Meadows and pastures 8.73 21.41 19.92 7.12 36.53 26.23 4.75 11.91 25.19 4.18

% Built 5.08 23.86 2.53 13.52 6.83 11.56 1.19 9.67 5.79 19.08

% Agricultural 7.99 29.09 27.04 58.86 73.35 33.94 3.68 55.38 6.20 28.41

     % Vegetable crops 1.87 5.94 2.52 18.22 0.60 7.03 0 0.05 1.87 5.94

     % Vineyards 0.06 3.30 0.40 0.45

     % Corn 1.8 2.12
A = non-exposed apiaries;    B = exposed apiaries;    * Calculated on the percentage of coverage;    ** Calculated on the number of polygons.

y = 0.2155x + 19.519
R² = 0.0374

0

10

20

30

40

50

60

70

80

0 20 40 60 80

Agricultural land coverage (ha)

C
u

m
u

la
ti

ve
 m

o
rt

al
it

y 
(%

)

Figure 3. Relationship between the cumulative beehive mortality and 
the agricultural land coverage.

0

5

10

15

20

25

30

35

40

C
u

m
u

la
ti

ve
 m

o
rt

al
it

y 
(%

)

Beehives with prevalence of 
natural habitats in the bu�ers

Natural Index > 1

Beehives with prevalence of 
anthropic habitats in the bu�ers

Natural Index < 1

Figure 4. Cumulative mortality in colonies placed in buffers at different 
Naturality Index values.



21Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Bellucci et al.  Honey bee health in Italy

A as well as apiary B within both the Dolomiti and 
Calanchi sites (score 5). The relationship between 
the beekeeper score and the winter and cumulative 
mortality indices, showed that the beekeepers’ 
skill was significantly related to both indices. The 
Spearman’s rank correlation coefficients showed a 
negative correlation between the beekeeper score 
and those two parameters, with values of ‑  0.7730 
and ‑  0.7722, respectively (p  =  0.009 in both cases). 
Two linear regressions showed that a one‑point 
increase in the beekeeper score corresponded to 
a 14% and 16% decrease in winter (Figure 5) and 
cumulative mortality (Figure 6), respectively.

Discussion
The weekly counts of dead adult honey bees in the 
under‑basket cages in our study were not related to 

Bee Virus was positively and significantly related 
with cumulative mortality (r

s
 = 0.6351, p = 0.048).

The diagnosis of Nosemosis was performed on 
64 honey bee samples. While N. ceranae was present 
in the samples from both A (average of 78.1%) and B 
(average of 65.6%) apiaries of the five NPAs, N. apis was 
never found (Table VIII). Statistical analysis showed 
that the frequency of N. ceranae in the A and B apiaries 
was not significantly different. The Spearman’s rank 
correlation coefficient used to measure correlation 
between the prevalence of N.  ceranae and the 
cumulative and winter mortality indices showed no 
significant correlation between them.

Table IX provides the results of the beekeeping 
skill assessment. The worst score for beekeepers’ 
management skill was given to Litorale B and 
Simbruini B (score 2) and the best was given to apiary 

Table VII. Average and standard deviation of heavy metal concentration detected (mg/kg) in each apiary of selected natural protected areas.

He
av

y 
m

et
al Dolomiti Calanchi Litorale San Rossore Simbruini

A B A B A B A B A B

Pb 0.042±0.021 0.045±0.032 0.042±0.027 0.045±00.041 0.033±0.015 0.035±0.020 0.038±0.028 0.053±0.019 0.033±0.036 0.053±0.042

Cu 0.369±0.402 0.551±0.436 0.178±0.035 0.165±0.075 0.567±0.461 0.304±0.288 0.204±0.082 0.150±0.058 0.183±0.075 0.299±0.218

Cd 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0 0.005±0

Cr 0.038±0.029 0.047±0.020 0.101±0.082 0.104±0.096 0.091±0.080 0.054±0.030 0.066±0.052 0.076±0.095 0.063±0.030 0.084±0.055
A = non-exposed apiaries;    B = exposed apiaries.

Table VIII. Pathogens detected in apiaries located in selected natural protected areas for the non-exposed and the exposed apiaries.

Dolomiti Calanchi Litorale San Rossore Simbruini Mean
A B A B A B A B A B A B

Varroa observations 
on adult honey bees

33% 33% 8% 13% 10% 5% 62% 47% 25% 20% 27.6% 23.6%

American Foul Brood
2/20 

(10%)
0/20 
(0%)

0/20 
(0%)

0/20 
(0%)

3/20 
(15%)

4/20 
(20%)

0/20 
(0%)

0/20 
(0%)

0/20 
(0%)

9/20 
(45%)

5/100 
(5%)

13/100 
(13%)

Acute Bee Paralysis 
Virus

0/7
(0%)

2/7 
(28.6%)

4/7 
(57.1%)

0/7
(0%)

7/7 
(100%)

7/7 
(100%)

3/5 
(60%)

4/5 
(80%)

2/6 
(33.3%)

3/7 
(42.8%)

16/32 
(50%)

16/33 
(48.5%)

Black Queen Cell Virus
0/7

(0%)
0/7

(0%)
1/7 

(14.3%)
0/7

(0%)
5/7 

(71.4%)
5/7 

(71,4%)
3/5 

(60%)
0/5

(0%)
0/6

(0%)
0/7

(0%)
9/32 

(28.1%)
5/33 

(15.2%)
Chronic Bee Paralysis 

Virus
6/7 

(85.7%)
0/7

(0%)
5/7 

(71.4%)
4/7 

(57.1%)
7/7 

(100%)
7/7 

(100%)
2/5 

(40%)
0/5

(0%)
1/6 

(16.7%)
6/7 

(85.7%)
21/32 

(65.6%)
17/33 

(51.5%)

Deformed Wings Virus
3/7 

(42.8%)
0/7

(0%)
4/7 

(57.1%)
5/7 

(71.4%)
7/7 

(100%)
6/7 

(85.7%)
4/5 

(80%)
4/5 

(80%)
6/6 

(100%)
6/7 

(85.7%)
24/32 
(75%)

21/33 
(63.6%)

Israeli Acute Paralysis 
Virus

0/7
(0%)

0/7
(0%)

0/7
(0%)

0/7
(0%)

0/7
(0%)

0/7
(0%)

0/5
(0%)

0/5
(0%)

0/6
(0%)

0/7
(0%)

0/32 
(0%)

0/33 
(0%)

Kashmir Bee Virus
0/7

(0%)
0/7

(0%)
0/7

(0%)
0/7

(0%)
2*/7 

(28.6%)
0/7

(0%)
0/5

(0%)
4*/5 

(80%)
0/6

(0%)
2*/7 

(28.6%)
2/32 

(6.3%)
6/33 

(18.2%)

Sac Brood Virus
5/7 

(71.4%)
5/7 

(71.4%)
6/7 

(85.7%)
1/7 

(14.3)
6/7 

(85.7%)
6/7 

(85.7%)
4/5 

(80%)
2/5 

(40%)
1/6 

(16.7%)
1/7 

(14.3%)
22/32 

(68.7%)
15/33 

(45.5%)

Nosema ceranae
7/7 

(100%)
7/7 

(100%)
6/7 

(85.7%)
3/7 

(42.9%)
7/7 

(100%)
5/7 

(71.4%)
1/5 

(20%)
2/5 

(40%)
4/6 

(66.7%)
4/6 

(66.7%)
25/32 

(78.1%)
21/32 

(65.6%)

Nosema apis
0/7

(0%)
0/7

(0%)
0/7

(0%)
0/7

(0%)
0/7

(0%)
0/7

(0%)
0/5

(0%)
0/5

(0%)
0/6

(0%)
0/6

(0%)
0/32 
(0%)

0/32 
(0%)

A = non-exposed apiaries;    B = exposed apiaries.



22 Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Honey bee health in Italy Bellucci et al.

et  al. 2013). A more accurate study is needed to 
substantiate the relationship between ABPV and KBV 
and colony mortality in combination with the varroa 
infestation level. The prevalence of N. ceranae did not 
significantly differ in A and B apiaries.

No PPP residues were found in this 1‑year study, 
excluding one case in which, during spring 2010, 
a low level of imidacloprid (0.0096 mg/kg) was 
found in a dead honey bee sample of apiary B at 
Calanchi. The active amount of imidacloprid found 
corresponded to about ¼ of the LD

50
 for honey bees. 

However, in that apiary, bee mortality did not exceed 
the mortality threshold in the under‑basket cages, 
and no abnormal mortality was found. It should be 
stressed that, in Italy, a rule1 (OJ of Italian Republic 
n. 221 of 20 September 2008) established an 
immediate precautionary suspension of the use of 
PPPs for seed dressing (but not other formulations, 
e.g., spray applications) when those PPPs contain 
any of several active substances such as clothianidin, 

the colony mortality observed in the five NPAs; this 
was probably due to  an absence of strong acute 
toxic effects caused by PPPs during the  year of 
monitoring activity.

The Kaplan‑Meier survival estimates (Figure  2) 
expressed mortality events more evident in exposed 
apiaries respect to non‑exposed apiaries during 
October/November, January/February, August. In 
these months, hive mortalities are usually related 
to V.  destructor. The Spearman’s rank correlation 
coefficient used to measure the relationship 
between severe V.  destructor infestations and the 
mortality indices showed no correlation between 
them. However, in acquiring this information, we 
should also consider that a more accurate method 
may be used to assess the level of varroa infestation; 
this would have to be applied to achieve a robust 
conclusion, avoiding the different interpretations of 
the four inspectors that evaluated the hives in the 
five NPAs. Indeed, the on‑field evaluation methods to 
detect the level of varroa infestation in adult honey 
bees, such as the use of icing sugar (Lee et al. 2010) 
or detergent solutions (Rinderer et  al. 2004), were 
still not in use at the time of our protocol definition.

While EFB was not found in the present study, 
AFB was found with a higher prevalence in apiary 
B (13%) respect to apiary A (5%) (Table VIII). The 
Spearman’s rank correlation coefficient did not 
highlight any correlation between the mean AFB 
prevalence and the mortality indices. At Simbruini B 
apiary, AFB caused the highest mortality with nine 
colonies affected (45%); this was caused by the poor 
awareness of the beekeeper. 

With regard to the honey bee viruses, ABPV was 
positively related to both winter and cumulative 
mortality, while KBV was positively related only to 
cumulative mortality. This could be explained by the 
prevalence of KBV, which appears to be higher in 
summer than in winter (Formato et  al. 2012, Cersini 

Table IX. Beekeepers’ honey bee management skill score in the 
investigated apiaries. Scores range from 1 (weak) to 5 (excellent).

NPA Apiary Beekeepers score

Dolomiti
A 5

B 5

Calanchi
A 5

B 5

San Rossore
A 3

B 3

Litorale
A 4

B 2

Simbruini
A 3

B 2
A = non-exposed apiaries;    B = exposed apiaries.

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Beekepers score

C
u

m
u

la
ti

ve
 m

o
rt

al
it

y 
(%

)

y = -15.638x + 84.362
R² = 0.5471

Figure 5. Relationship between beekeeper management skill score and 
cumulative mortality (%).

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6

Beekepers score

W
in

te
r 

m
o

rt
al

it
y 

(%
)

y = -13.652x + 73.014
R² = 0.5243

Figure 6. Relationship between beekeeper management skill score and 
winter mortality (%).

1  Decreto dirigenziale. 2008. Sospensione cautelativa dell'autorizzazione 
di impiego per la concia di sementi, dei prodotti fitosanitari contenenti 
le sostanze attive clothianidin, thiamethoxam, imidacloprid e fipronil, 
ai sensi dell'articolo 13, comma 1, del decreto del Presidente della 
Repubblica 23 aprile 2001, n. 290. OJ, 221 of the 20.09.2008.



23Veterinaria Italiana 2019, 55 (1), 15‑25. doi: 10.12834/VetIt.1209.6739.4

Bellucci et al.  Honey bee health in Italy

the A and B apiaries. In fact, heavy metals do 
not decompose and are easily transported at 
considerable distances by air currents, being spread 
out in an area independently from their natural, 
rural, urban, or industrial characteristics (Devillers 
et al. 2002).

Finally, the managerial skills of beekeepers were 
significantly related both to the winter and 
cumulative mortality, confirming that beekeeping 
management, like honey bee pathogens and 
environmental pollution, can contribute to threaten 
the health of honey bee colonies (Oldroyd 2007; 
vanEngelsdorp et al. 2008).

Conclusions
Our monitoring activity did not reveal a significant 
effect on colony mortality caused by either 
anthropogenic pollutants or honey bee pathogens 
that were observed in all the monitored apiaries in 
different proportions, even if there was a significant 
increase in risk in exposed areas (B apiaries) 
compared to unexposed ones (A apiaries). The 
results demonstrate that the application of the 
under‑basket cage criterion used to evaluate colony 
mortality is not effective in apiaries that are not 
involved in acute toxic effects (e.g., acute toxic PPP 
effects). The relation between honey bee mortality 
and Naturality index is interestingly observed 
even in the context in which the present study 
was conducted, i.e. Natural Protected Areas. In the 
areas most heavily affected by the colony mortality 
(Litorale, San Rossore, and Simbruini), the poor 
colony management skills of the beekeepers played 
the most important role in colony losses. 

This study confirms previous studies showing that 
colony collapse is the consequence of the interaction 
of multiple factors, both natural and anthropogenic, 
that affect honey bee health.

fipronil, imidacloprid, and thiamethoxam. As a 
consequence, since 2009 during corn sowing season, 
no neonicotinoid‑dressed seeds were allowed in Italy 
and only two honey bee mortality outbreaks related 
to neonicotinoid‑dressed seeds were recorded in 
that year in Italy. For this reason, the ban was then 
extended until June 2013, and no further cases of 
this type of mortality have been reported. It could 
have somehow reduced the possibility of detecting 
neonicotinoid residues in the investigated matrices, 
despite the permitted use of neonicotinoids on fruit 
trees, vineyards, and other shrubs. 

In NPAs, the vegetation coverage resulted related 
with the health status of colonies, even if it is not clear 
the influence of intensive agricultural techniques. 
However, it has to be considered that, within the 
NPAs, large areas of industrial or agricultural lands 
were not usually present. The failure to find a direct 
link between colony mortality and the crop coverage 
may be caused by the heterogeneity of the farming 
procedures used in the study area (e.g., the diversity 
of the treatments and cropping systems employed). 
Despite the presence of large areas of farmland, 
vegetable crops, and vineyards, the low colony 
mortality recorded in both types of apiaries of 
Calanchi NPA, should be attributed to the spreading 
of sustainable and organic agricultural production 
promoted by the park authorities (Naylor and Ehrlich 
1997), as well as to the apiary management skills of 
highly professional beekeepers..

The colonies of the apiaries B in Litorale and San 
Rossore Parks, showed significantly higher mortality 
compared to the associated apiaries A. In both of 
these NPAs, some factors could have influenced this 
mortality trend, such as the presence of intensive 
horticultural crops (Litorale), an autumn weed 
control treatment with herbicides (San Rossore), and 
the presence of an airport (Litorale).

The heavy metal concentrations detected in the 
honey samples did not statistically differ between 



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