Natural immunity in conventionally and organically reared turkeys and its relation with antimicrobial resistance.

Suboptimal animal welfare may affect natural immunity, rendering animals more susceptible to environmentally conditioned diseases, including those requiring antimicrobial treatment, which may promote antimicrobial resistance (AMR) in bacterial populations. Herewith, we tested the hypothesis that conventionally raised turkeys have higher levels of AMR in indicator Escherichia coli bacteria, but lower levels of natural immunity, as compared to turkeys reared under organic conditions. Litter and serum samples were collected from 28 conventional and 4 organic turkey farms: E. coli isolates from litter were tested for resistance to 14 antimicrobials, while 3 parameters of natural immunity (i.e., lysozyme, hemolytic complement levels, and serum bactericidal activity) were assessed in the sera. Resistant E. coli isolates were identified in both conventional and organic farms but generally more frequently in conventional farms. High rates of resistance to ampicillin (96%), tetracycline (95%), streptomycin (82%), sulfamethoxazole (80%), ciprofloxacin (73%), and trimethoprim (71%), as well as high rates of multiresistance, were observed in conventional farms. Organically raised turkeys had significantly higher levels of lysozyme and serum bactericidal activity than conventional turkeys, and these levels were also higher in turkeys housed in farms where AMR frequency was lower. Findings support the hypothesis that conventional farming conditions may affect turkeys' natural immunity, rendering the animals more susceptible to environmentally conditioned diseases requiring antimicrobial treatment, which would in turn promote AMR. Reducing AMR in turkey farming is therefore more likely to be successful when considering animal welfare as an option to reduce the need of antimicrobial use.

Positive Reinforcement-Based Training for Self-Loading of Meat Horses Reduces Loading Time and Stress-Related Behavior.

The present work aimed to evaluate the efficacy of a self-loading training using positive reinforcement on stress-related behaviors shown by meat horses during loading procedures into a truck. Thirty-two meat horses (M = 18; F = 14; 6 month-old) were included in the study. All horses had limited interactions with the farmer and were not used to be restrained nor lead by halter. Horses were divided in two groups: Control Group (C; N = 14) and Training Group (T; N = 18). T horses were trained to self-load: in order to teach the horses to enter into the truck, a targeting training technique throughout a shaping process was applied. Training sessions were performed three times a week, from 9:30 a.m. to 1:30 p.m. and from 2:30 p.m. to 4:30 p.m., for 6 weeks; training was then repeated once a week to maintain the memory until the transport toward a slaughterhouse. The loading phase was video-recorded and loading time was directly recorded using a stopwatch. All horses were transported to the same slaughterhouse in 14 different days using the same truck. Behavior was subsequently analyzed with a focal animal continuous recording method. Loading time was shorter in T horses (mean ± SD = 44.44 ± 47.58 s) than in C horses (mean ± SD = 463.09 ± 918.19 s) (T-test; p = 0.019). T horses showed more forward locomotion toward the truck than C horses (T-test; p = 0.029). Our preliminary findings suggest that self-loading training may be useful to mitigate loading-related stress in meat horses, minimally socialized with humans.

Trends and correlates of antimicrobial use in broiler and turkey farms: a poultry company registry-based study in Italy.

BACKGROUND: Antimicrobial usage (AMU) in livestock plays a key role in the emergence and spread of antimicrobial resistance. Analysis of AMU data in livestock is therefore relevant for both animal and public health. OBJECTIVES: To assess AMU in 470 broiler and 252 turkey farms of one of Italy's largest poultry companies, accounting for around 30% of national poultry production, to identify trends and risk factors for AMU. METHODS: Antimicrobial treatments administered to 5827 broiler and 1264 turkey grow-out cycles in 2015-17 were expressed as DDDs for animals per population correction unit (DDDvet/PCU). A retrospective analysis was conducted to examine the effect of geographical area, season and prescribing veterinarian on AMU. Management and structural interventions implemented by the company were also assessed. RESULTS: AMU showed a 71% reduction in broilers (from 14 to 4 DDDvet/PCU) and a 56% reduction in turkeys (from 41 to 18 DDDvet/PCU) during the study period. Quinolones, macrolides and polymyxins decreased from 33% to 6% of total AMU in broilers, and from 56% to 32% in turkeys. Broiler cycles during spring and winter showed significantly higher AMU, as well as those in densely populated poultry areas. Different antimicrobial prescribing behaviour was identified among veterinarians. CONCLUSIONS: This study evidenced a decreasing trend in AMU and identified several correlates of AMU in broilers and turkeys. These factors will inform the design of interventions to further reduce AMU and therefore counteract antimicrobial resistance in these poultry sectors.

Mycobacterium caprae in a dairy farm in Northeast Italy.

Veneto region, Northeast Italy, has been declared officially free from bovine tuberculosis since 2008, although the disease is sporadically detected in association with cattle trade. In September 2015, bovine tuberculosis was detected in a dairy cattle farm of the region, in a holding with 69 animals. The herd underwent single intradermal tuberculin testing as part of the regional surveillance plan, and 24 animals resulted positive. Mycobacterium caprae was evidenced in 22 samples, further genotyped by PCR-based assays, as Allgäu type. Epidemiological investigation reported that sixteen animals were introduced from an officially tuberculosis free Member State in previous years. Nevertheless, spoligotyping and multilocus variable tandem repeat analysis (MLVA) indicated that M. caprae was strictly related to the strain circulating in 2007-2009 in Trento province, although no at-risk contacts were described. M. caprae is a zoonotic pathogen and further analyses are warranted in order to control its spread and impact on public health and animal trade.

Impact of pre-slaughter transport conditions on stress response, carcass traits, and meat quality in growing rabbits.

In growing rabbits (n = 320; 84 d of age), an increase in the journey duration to the slaughterhouse from 1 h to 3 h under mild climatic conditions (10-13 °C; 75-90% relative humidity) affected several stress indicators in the plasma and muscle collected at slaughter (cortisol; corticosterone; lactate dehydrogenase, LDH; lactate; heat shock protein 70 kDa, HSP70; osmolality, and Na) (0.001 < P < .05), reduced muscle L*, b* (P < .01), and meat shear force (P < .05), and increased the dressing out percentage and muscle pH (P < .01). An increase in the lairage duration before slaughtering from 30 min to 3 h affected muscle cortisol and plasma creatinine kinase, LDH, lactate, and HSP70 (0.001 < P < .05), increased dressing out percentage and muscle pH (P < .05), but decreased meat shear force (P < .001). The interaction between journey and lairage duration was significant for several stress indicators and meat quality.

Association between passive immunity and health status of dairy calves under 30 days of age.

Calves are highly susceptible to disease and mortality occurrence within the first month of life. Even if failed transfer of passive immunity (FTPI) is commonly recognized as a main factor affecting calf health and survival, conflicting results are reported in literature about the association between passive immunity (PI) and calf health, especially regarding enteric diseases. Therefore, a prospective cohort study was conducted on 78 calves of three Italian dairy farms during winters of years 2014-2016, with the specific aim of evaluating the association between PI and health status of calves within 30 days of age under field conditions. Blood samples were collected between 1 and 5 days of age from each calf included in the study, and disease and mortality occurrence was monitored throughout the first month of life. Additionally, fecal samples were collected from calves with scours before treatment. Blood serum samples were tested by an electrophoretic method for the assessment of immunoglobulin (Ig) concentration, whereas fecal samples were submitted to ELISA test for positivity to Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium spp. Only enteric diseases occurred in calves of this study. Calves that suffered from diarrhea or died within the first month of life had lower serum Ig concentrations than those that remained healthy or survived (P < .05). Even if not significantly (P = .127), lower serum Ig concentrations were observed in sick calves that had been treated with antibiotics compared to those that had not been treated. The odds of disease and mortality occurrence were 24 (95% CI = 3-231) and 11 (95% CI = 1-111) times higher, respectively, for calves with FTPI (serum Ig concentration <10.0 g/L) than for those with an adequate PI transfer (P <  .05). Calves with adequate PI transfer had also a 6-day delay in the age at first disease onset compared to those with FTPI (P < .01). Even if estimated on a small number of calves, those with FTPI had higher risks of enteric infections by rotavirus (odds ratio = 12; 95% CI = 1-137) and Cryptosporidium spp. (odds ratio = 9; 95% CI = 1-72) (P < .05). In this study, the PI level influenced the occurrence of enteric diseases and mortality in calves under one month of age, confirming the importance of a proper colostrum provision to calf health and, consequently, to the reduction of antimicrobial use in dairy farming. However, further investigations are needed, particularly focusing on the relationship between PI and specific enteropathogen infections in calves.

Proposal for a new subtype of the zoonotic genotype 3 Hepatitis E virus: HEV-3l.

The near-complete genomic sequences of two hepatitis E virus (HEV) strains, detected from feces of infected pigs, were obtained. Phylogenetic analysis and p-distance comparisons of the complete coding regions showed a close relationship to the French swine strain FR-SHEV3c-like detected in 2006 (p-distance value 0.101), belonging to HEV-3 but not assigned to any known subtype. The three HEV sequences showed, relatively high nucleotide distances (p-distance >0.129) compared to the other defined HEV subtype references and unclassified strains. The HEV classification criteria and the high sequence similarity suggest that these strains can be assigned to a putative novel subtype of genotype 3, HEV-3l.

Genetic Diversity of Highly Pathogenic Avian Influenza A(H5N8/H5N5) Viruses in Italy, 2016-17.

In winter 2016-17, highly pathogenic avian influenza A(H5N8) and A(H5N5) viruses of clade 2.3.4.4 were identified in wild and domestic birds in Italy. We report the occurrence of multiple introductions and describe the identification in Europe of 2 novel genotypes, generated through multiple reassortment events.

The effect of crate height on the behavior of female turkeys during commercial pre-slaughter transportation.

Limited information is available on suitable height of transport crates for turkeys. We compared behaviors and physiological indicators of four groups of 10 female turkeys each confined in either conventional (38.5 cm height) or experimental (77 cm height) crates during six commercial pre-slaughter transportations for 86 km (76 ± 4 min) along two tracts with one-lane streets, crossroads, bends, roundabouts (S1 and S2) and a highway tract (H) between S1 and S2. Only 36% of birds in the higher crates maintained a standing position. In conventional versus experimental crates, the frequency of rising attempts was five/bird/hour versus less than one/bird/hour, while wing flapping was seven/bird/hour versus 20/bird/hour, and balance loss was one versus four/bird/hour. The behaviors of both groups differed significantly according to the route tract, with a lower frequency of stress-related behaviors at H. No scratches, fractures or hematomas were detected in any birds after transportation. Crate height had no significant effect on hemato-biochemical markers. These results suggest that crates enabling a standing position may increase potentially dangerous behaviors. Moreover, busy and curvy routes should be avoided, as they may contribute to increasing the frequency of stress-related behaviors.

Circulation of multiple genotypes of H1N2 viruses in a swine farm in Italy over a two-month period.

In August 2012 repeated respiratory outbreaks caused by swine influenza A virus (swIAV) were registered for a whole year in a breeding farm in northeast Italy that supplied piglets for fattening. The virus, initially characterized in the farm, was a reassortant Eurasian avian-like H1N1 (H1avN1) genotype, containing a haemagglutinin segment derived from the pandemic H1N1 (A(H1N1)pdm09) lineage. To control infection, a vaccination program using vaccines against the A(H1N1)pdm09, human-like H1N2 (H1huN2), human-like H3N2 (H3N2), and H1avN1 viruses was implemented in sows in November 2013. Vaccine efficacy was assessed by sampling nasal swabs for two months in 35-75 day-old piglets born from vaccinated sows. Complete genome sequencing of eight swIAV-positive nasal swabs collected longitudinally from piglets after the implementation of the vaccination program was conducted to investigate the virus characteristics. Over the two-month period, two different genotypes involving multiple reassortment events were detected. The unexpected circulation of multiple reassortant genotypes in such a short time highlights the complexity of the genetic diversity of swIAV and the need for a better surveillance plan, based on the combination of clinical signs, epidemiological data and whole genome characterization.

Unexpected Interfarm Transmission Dynamics during a Highly Pathogenic Avian Influenza Epidemic.

UNLABELLED: Next-generation sequencing technology is now being increasingly applied to study the within- and between-host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep-sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel (i) the epidemic virus population diversity, (ii) the evolution of virus pathogenicity, and (iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the cocirculation in the index case of two viral strains showing a different insertion at the hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess interfarm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed interfarm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain. IMPORTANCE: The within- and between-host evolutionary dynamics of a highly pathogenic avian influenza (HPAI) strain during a naturally occurring epidemic is currently poorly understood. Here, we perform for the first time an in-depth sequence analysis of all the samples collected during a HPAI epidemic and demonstrate the importance to complement outbreak investigations with genetic data to reconstruct the transmission dynamics of the viruses and to evaluate the within- and between-farm genetic diversity of the viral population. We show that the evolutionary transition from the low pathogenic form to the highly pathogenic form occurred within the first infected flock, where we identified haplotypes with hemagglutinin cleavage site of different lengths. We also identify the index case as the major source of virus, indicating that prompt application of depopulation measures is essential to limit virus spread to other farms.

West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Productive and reproductive performances of dairy cattle herds in Treviso province, Italy (2009-2012): an assessment of the potential impact of Schmallenberg virus epidemic.

BACKGROUND: Schmallenberg virus (SBV) has spread across Europe since mid-2011, causing unspecific and transitory symptoms in ruminants and congenital malformations in their offspring. Evidence for the impact of SBV on cattle (re)productive performance is limited. Using a comprehensive data set from a SBV-affected province in North-East Italy, this study aimed at assessing the potential impact of SBV emergence on 11 productive and reproductive performance indicators of dairy cattle herds, accounting for weather conditions and other herd-level factors that could also influence these indicators. RESULTS: A total of 127 farms with an average of 71 cows per farm (range 29-496) were monitored monthly from January 2009 to June 2012. Mixed-effects linear models for longitudinal data were used to assess the average variation in herds' performance indicators over semesters (Jan-Jun 2009, Jul-Dec 2009, Jan-Jun 2010, Jul-Dec 2010, Jan-Jun 2011, Jul-Dec 2011, Jan-Jun 2012) and trimesters therein. Taking the second semester of 2011 as reference, significant decreases in the average lactation length (-6 days, on average) and calving-to-conception interval (-4 days, on average) were observed relative to the same semesters of the years 2010 and 2009, respectively. Similarly, during the last trimester of 2011, which is most likely to cover the SBV infection period in the study area, there was an average decrease of -4 days (lactation length) and -7 days (calving-to-conception interval) compared to the same trimesters of the years 2010 and 2009, respectively. However, the observed decreases actually represent a positive outcome that is not as such imputable to SBV emergence, but rather reflects other beneficial changes in farm management. None of the other indicators showed significant variations, confirming the relatively mild expression of SBV infection in cattle. CONCLUSIONS: Although the emergence of SBV might have significantly affected the (re)productive performance of some individual farms, we concluded that overall at the province level there were no significant variations attributable to SBV, at least not in a way that would lead to negative effects on farm profitability.

The degree of acceptability of swine blood values at increasing levels of hemolysis evaluated through visual inspection versus automated quantification.

The pronounced fragility that characterizes swine erythrocytes is likely to produce a variable degree of hemolysis during blood sampling, and the free hemoglobin may then unpredictably bias the quantification of several analytes. The aim of this study was to evaluate the degree of acceptability of values obtained for several biochemical parameters at different levels of hemolysis. Progressively increased degrees of physical hemolysis were induced in 3 aliquots of 30 nonhemolytic sera, and the relative effects on the test results were assessed. To define the level of hemolysis, we used both visual estimation (on a scale of 0 to 3+) and analytical assessment (hemolytic index) and identified the best analytical cutoff values for discriminating the visual levels of hemolysis. Hemolysis led to a variable and dose-dependent effect on the test results that was specific for each analyte tested. In mildly hemolyzed specimens, C-reactive protein, haptoglobin, ß1-globulin, ß2-globulin, α1-globulin, γ-globulin, sodium, calcium, and alkaline phosphatase were not significantly biased, whereas α2-globulin, albumin, urea, creatinine, glucose, total cholesterol, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, nonesterified fatty acids, bilirubin, phosphorus, magnesium, iron, zinc, copper, lipase, triglycerides, lactate dehydrogenase, unbound iron-binding capacity, and uric acid were significantly biased. Chloride and total protein were unbiased even in markedly hemolyzed samples. Analytical interference was hypothesized to be the main source of this bias, leading to a nonlinear trend that confirmed the difficulty in establishing reliable coefficients of correction for adjusting the test results.

Control of a Reassortant Pandemic 2009 H1N1 Influenza Virus Outbreak in an Intensive Swine Breeding Farm: Effect of Vaccination and Enhanced Farm Management Practices.

Influenza A viruses in swine cause considerable economic losses and raise concerns about their zoonotic potential. The current paucity of thorough empirical assessments of influenza A virus infection levels in swine herds under different control interventions hinders our understanding of their effectiveness. Between 2012 and 2013, recurrent outbreaks of respiratory disease caused by a reassortant pandemic 2009 H1N1 (H1N1pdm) virus were registered in a swine breeding farm in North-East Italy, providing the opportunity to assess an outbreak response plan based on vaccination and enhanced farm management. All sows/gilts were vaccinated with a H1N1pdm-specific vaccine, biosecurity was enhanced, weaning cycles were lengthened, and cross-fostering of piglets was banned. All tested piglets had maternally-derived antibodies at 30 days of age and were detectable in 5.3% of ~90 day-old piglets. There was a significant reduction in H1N1pdm RT-PCR detections after the intervention. Although our study could not fully determine the extent to which the observed trends in seropositivity or RT-PCR positivity among piglets were due to the intervention or to the natural course of the disease in the herd, we provided suggestive evidence that the applied measures were useful in controlling the outbreak, even without an all-in/all-out system, while keeping farm productivity at full.

Evolutionary trajectories of two distinct avian influenza epidemics: Parallelisms and divergences.

Influenza A virus can quickly acquire genetic mutations that may be associated with increased virulence, host switching or antigenic changes. To provide new insights into the evolutionary dynamics and the adaptive strategies of distinct avian influenza lineages in response to environmental and host factors, we compared two distinct avian influenza epidemics caused by the H7N1 and H7N3 subtypes that circulated under similar epidemiological conditions, including the same domestic species reared in the same densely populated poultry area for similar periods of time. The two strains appear to have experienced largely divergent evolution: the H7N1 viruses evolved into a highly pathogenic form, while the H7N3 did not. However, a more detailed molecular and evolutionary analysis revealed several common features: (i) the independent acquisition of 32 identical mutations throughout the entire genome; (ii) the evolution and persistence of two sole genetic groups with similar genetic characteristics; (iii) a comparable pattern of amino acid variability of the HA proteins during the low pathogenic epidemics; and (iv) similar rates of nucleotide substitutions. These findings suggest that the evolutionary trajectories of viruses with the same virulence level circulating in analogous epidemiological conditions may be similar. In addition, our deep sequencing analysis of 15 samples revealed that 17 of the 32 parallel mutations were already present at the beginning of the two epidemics, suggesting that fixation of these mutations may occur with different mechanisms, which may depend on the fitness gain provided by each mutation. This highlighted the difficulties in predicting the acquisition of mutations that can be correlated to viral adaptation to specific epidemiological conditions or to changes in virus virulence.

Emergence of a highly pathogenic avian influenza virus from a low-pathogenic progenitor.

UNLABELLED: Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low-pathogenic (LPAI) progenitor viruses currently is poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultradeep-sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high- and low-pathogenicity viruses undertaken to date and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties. IMPORTANCE: The Italian H7 AI epidemic of 1999 to 2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP forms from LP precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data, and ultradeep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low-frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.

Environmental correlates of H5N2 low pathogenicity avian influenza outbreak heterogeneity in domestic poultry in Italy.

Italy has experienced recurrent incursions of H5N2 avian influenza (AI) viruses in different geographical areas and varying sectors of the domestic poultry industry. Considering outbreak heterogeneity rather than treating all outbreaks of low pathogenicity AI (LPAI) viruses equally is important given their interactions with the environment and potential to spread, evolve and increase pathogenicity. This study aims at identifying potential environmental drivers of H5N2 LPAI outbreak occurrence in time, space and poultry populations. Thirty-four environmental variables were tested for association with the characteristics of 27 H5N2 LPAI outbreaks (i.e. time, place, flock type, number and species of birds affected) occurred among domestic poultry flocks in Italy in 2010-2012. This was done by applying a recently proposed analytical approach based on a combined non-metric multidimensional scaling, clustering and regression analysis. Results indicated that the pattern of (dis)similarities among the outbreaks entailed an underlying structure that may be the outcome of large-scale, environmental interactions in ecological dimension. Increased densities of poultry breeders, and increased land coverage by industrial, commercial and transport units were associated with increased heterogeneity in outbreak characteristics. In areas with high breeder densities and with many infrastructures, outbreaks affected mainly industrial turkey/layer flocks. Outbreaks affecting ornamental, commercial and rural multi-species flocks occurred mainly in lowly infrastructured areas of northern Italy. Outbreaks affecting rural layer flocks occurred mainly in areas with low breeder densities in south-central Italy. In savannah-like environments, outbreaks affected mainly commercial flocks of galliformes. Suggestive evidence that ecological ordination makes sense genetically was also provided, as virus strains showing high genetic similarity clustered into ecologically similar outbreaks. Findings were informed by hypotheses about how ecological interactions among poultry populations, viruses and their environments can be related to the observed patterns of H5N2 LPAI occurrence. This may prove useful in enhancing future interventions by developing site-specific, ecologically-grounded strategies.

Determinants of the population growth of the West Nile virus mosquito vector Culex pipiens in a repeatedly affected area in Italy.

BACKGROUND: The recent spread of West Nile Virus in temperate countries has raised concern. Predicting the likelihood of transmission is crucial to ascertain the threat to Public and Veterinary Health. However, accurate models of West Nile Virus (WNV) expansion in Europe may be hampered by limited understanding of the population dynamics of their primary mosquito vectors and their response to environmental changes. METHODS: We used data collected in north-eastern Italy (2009-2011) to analyze the determinants of the population growth rate of the primary WNV vector Culex pipiens. A series of alternative growth models were fitted to longitudinal data on mosquito abundance to evaluate the strength of evidence for regulation by intrinsic density-dependent and/or extrinsic environmental factors. Model-averaging algorithms were then used to estimate the relative importance of intrinsic and extrinsic variables in describing the variations of per-capita growth rates. RESULTS: Results indicate a much greater contribution of density-dependence in regulating vector population growth rates than of any environmental factor on its own. Analysis of an average model of Cx. pipiens growth revealed that the most significant predictors of their population dynamics was the length of daylight, estimated population size and temperature conditions in the 15 day period prior to sampling. Other extrinsic variables (including measures of precipitation, number of rainy days, and humidity) had only a minor influence on Cx. pipiens growth rates. CONCLUSIONS: These results indicate the need to incorporate density dependence in combination with key environmental factors for robust prediction of Cx. pipiens population expansion and WNV transmission risk. We hypothesize that detailed analysis of the determinants of mosquito vector growth rate as conducted here can help identify when and where an increase in vector population size and associated WNV transmission risk should be expected.

Ecological niche modelling of potential West Nile virus vector mosquito species and their geographical association with equine epizootics in Italy.

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.