A non-invasive method to monitor farmland bird exposure to triazole fungicides.

The treatment of seeds with pesticides is an extended practice in current agriculture. There is a high risk of exposure in granivorous birds, such as the red-legged partridge (Alectoris rufa), that can consume those seeds remaining on the surface during sowing. Fungicide exposure could in turn affect bird reproductive capacity. To better understand to what extent triazole fungicides are a threat to granivorous birds, we need an easy and reliable method to quantify field exposure. In this study, we tested a novel non-invasive method to detect the presence of triazole fungicide residues in farmland bird faeces. We experimentally exposed captive red-legged partridges to validate the method, and then applied it in a real scenario to assess exposure of wild partridges. We exposed adult partridges to seeds treated with two formulations containing triazole fungicides as active ingredients: Vincit®Minima (flutriafol 2.5%) and Raxil®Plus (prothioconazole 25% and tebuconazole 15%). We collected two types of faeces (caecal and rectal samples) immediately after exposure and 7 days later and quantified the concentrations of the three triazoles and their common metabolite (1,2,4-triazole). The three active ingredients and 1,2,4-triazole were only detected in faeces collected immediately after exposure. Triazole fungicide detection rates in rectal stool were 28.6%, 73.3% and 80% for flutriafol, prothioconazole and tebuconazole, respectively. In caecal samples, detection rates were 40%, 93.3% and 33.3%, respectively. 1,2,4-triazole was detected in 53% of rectal samples. For an applied use of the method in the field, we collected 43 faecal samples from wild red-legged partridges during autumn cereal seed sowing and found detectable levels of tebuconazole in 18.6% of the analysed wild partridges. The results of the experiment were then used to estimate actual exposure levels from this prevalence value found in wild birds. Our study shows that faecal analysis can be a useful tool to assess farmland bird exposure to triazole fungicides, when samples are fresh and the method has been validated for the detection of target molecules.

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Bird exposure to fungicides through the consumption of treated seeds: A study of wild red-legged partridges in central Spain.

Sown seeds are a key component of many farmland birds' diets due to natural food shortages in autumn and winter. Because these seeds are often treated with pesticides, their ingestion by birds can result in toxic effects. For risk assessment, data on treated seed toxicity should be combined with information about exposure risk for wild birds and the factors that modulate it. We characterized the exposure of red-legged partridges to pesticide-treated seeds through the analysis of digestive contents of birds shot by hunters (n = 194) in an agricultural region in central Spain. We measured the contribution of sown seeds to the partridges' diet and how it related to pesticide exposure. Moreover, we evaluated the influence of landscape composition on the intake of sown seeds and pesticides by partridges. During peak sowing time, seeds constituted half (50.7%) of the fresh biomass ingested by partridges, which consumed mostly winter cereal seeds (42.3% of biomass). Residues of seven fungicides and one insecticide (active ingredients) were detected in 33.0% of birds. The presence of pesticides in digestive contents was linked to the ingestion of cereal sown seeds. Moreover, dietary exposure of birds to pesticides was modulated by landscape characteristics, being lower in areas with heterogeneous landscapes, greater habitat mosaic and more natural vegetation. The estimated dietary intake of pesticides resulting from our field observations, in combination with experimental data on pesticide toxicity, raise concerns about the risks that pesticide-treated cereal seeds pose to granivorous bird populations. Our results highlight the importance of farming landscape composition and diversification, which should be considered as a priority in the agricultural policy to mitigate pesticide risks to farmland birds through the consumption of treated seeds.

Farmland composition and farming practices explain spatio-temporal variations in red-legged partridge density in central Spain.

Many farmland bird populations are declining, and their negative trends are often associated with changes in land-use or farming practices, including the use of agrochemicals. The red-legged partridge (RLP) is a Mediterranean farmland game species of high socio-economic importance whose populations are thought to have declined sharply since the mid-20th century associated with farmland changes. However, no large-scale studies have tested whether abundance or trends of RLP are related to farmland composition or management. We used hierarchical distance sampling models to estimate RLP abundance in 2010 in central Spain (Castilla-La Mancha), a main European population stronghold of this species. We studied associations between RLP density and land-uses (including variation in management: irrigated crops or organic farming). We also assessed regional abundance variation over seven years (2010-2017) and its relationship with changes in land-use. Our results show that RLP abundance increased with the availability of natural vegetation and traditional rain-fed vineyards, but decreased with increasing proportions of tree crops and irrigated vineyards; the latter association was less pronounced in areas sensitive to nitrate contamination in water, where the amount of fertilizers applied in farmland and use of certain farming practices is more strictly regulated. These results support the idea that increases in intensive vineyards are detrimental to the RLP. We also report a strong population decline of RLP in the region, with a 51% abundance reduction in seven years. This decline was steeper in areas where more natural vegetation had been lost and where ecological tree crops had increased. Overall, our results indicate that changes in land-use (type of crop, or the destruction of natural vegetation in farmland) and farming practices (e.g. use of irrigation in certain crops, use of nitrates) have important impacts on this farmland bird, affecting both spatial distribution and population dynamics.

Genome-wide markers redeem the lost identity of a heavily managed gamebird.

Heavily managed wildlife may suffer from genetic homogenization and reshuffling of locally adapted genotypes with non-native ones. This phenomenon often affects natural populations by reducing their evolutionary potential and speeding up the ongoing biodiversity crisis. For decades, the red-legged partridge (Alectoris rufa), an intensively managed gamebird of conservation concern and considerable socio-economic importance, has been subjected to extensive releases of farm-reared hybrids with the chukar partridge (Alectoris chukar) and translocations irrespective of subspecific affinity. These practices have led to serious concerns that the genetic integrity and biogeographic structure of most red-legged partridge populations are irreversibly affected, as suggested by previous studies based on few genetic markers. Using over 168 000 genome-wide loci and a sampling across the entire A. rufa range, we detected unexpectedly limited and spatially uneven chukar introgression as well as significant intraspecific structure. We demonstrate that species widely feared to have irretrievably lost their genetic identity are likely to be much less affected by unsuitable management practices than previously assumed. Our results spell the need for a radical re-think on animal conservation, possibly restoring native status to populations long treated as compromised. Our study exemplifies how the application of innovative conservation-genomic methods is key to solving wildlife management problems dealing with introgressive hybridization worldwide.

An open platform system based on SNP type genetic markers for discrimination between Alectoris rufa and Alectoris chukar.

The red-legged partridge (Alectoris rufa) is one of the most emblematic game species in Southern Europe. For the conservation of its natural populations against hybridization with chukar partridges (Alectoris chukar) a public and agreed control system able to detect genetic introgression between the two species should be established. As the already available method has not been implemented yet, this paper presents an improvement of the genetic analysis technique by using an open platform system to optimize the diagnostic procedure. Here we present the results obtained from the design of an Open Array™ platform with the available SNPs with proved diagnosis capacity between the two species of interest. By this procedure we genotyped 380 partridge samples, both from farms and field populations, which resulted in an overall percentage of genotyping performed with success of 99.64%. The Open Array genotyping plates showed high performance, specificity and an easy reproducibility compared to conventional techniques of genotyping.

Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge.

Triazole fungicides are the most widely used products to treat cereal seeds. Granivorous birds, such as red-legged partridges (Alectoris rufa), which consume seeds left on the surface of fields after sowing, have a high risk of exposure. As triazole fungicides can affect sterol synthesis, we tested the hypothesis that treated seed consumption could alter the synthesis of sex hormones and reduce the reproductive capacity of partridges. We exposed adult partridges to seeds treated with four different formulations containing triazoles as active ingredients (flutriafol, prothioconazole, tebuconazole, and a mixture of the latter two) simulating a field exposure during the late autumn sowing season. All treatments produced biochemical changes and an overexpression of genes encoding for enzymes involved in the biosynthesis of sterols and steroid hormones, such as PMVK, ABCA1, MVD, PSCK9, DHCR7 and HSD17B7. Plasma levels of oestradiol were reduced in partridges exposed to tebuconazole. We also monitored reproduction 3 months after exposure (laying date, egg fertilization and hatching rates). We observed a 14-day delay in the laying onset of partridges that had been exposed to flutriafol as compared to controls. These results show that the consumption of seeds treated with triazole fungicides has the potential to affect granivorous bird reproduction. We recommend the evaluation of lagged reproductive effects as part of the protocols of environmental risk assessment of pesticides in wild birds in light of the effects resulting from the exposure to triazole-treated seeds.

Protozoan and Helminth Infections of Aviary-Reared Alectoris rufa (Galliformes: Phasianidae) before Releasing for Hunting in the Czech Republic: Infection Dynamics and Potential Risks.

Age-related dynamics of protozoan and helminth infections in the red-legged partridge, Alectoris rufa, were studied in an intensive breeding aviary in the Czech Republic before releasing birds for hunting purposes. Pooled fecal samples (n = 900) were examined over 3 rearing seasons (2012-2014). A total of 4 protozoan species, Cryptosporidium baileyi, Cryptosporidium meleagridis, Eimeria kofoidi, and Eimeria legionensis, and 4 helminths, Capillaria phasianina, Eucoleus perforans, Heterakis gallinarum, and Syngamus trachea, was found. The most common parasite was S. trachea (prevalence 20-26%) in dead birds (n = 99), which represents a high risk for breeders. Co-occurrence of protozoans and helminths indicated similarities in infection dynamics throughout the 3 breeding seasons. Mixed infections of Cryptosporidium baileyi and C. meleagridis with other parasitic species are reported for the first time. Our findings provide new insights into breeding of A. rufa and may help to improve the efficacy of disease control strategies and prevention, especially with the potential for spreading of parasitic infections to wildlife through released birds into open areas.

Haemoparasites in red-legged partridge (Alectoris rufa): first record of Haemoproteus sp. in Italy?

During a health survey in the Province of Alessandria (NW Italy) 267 free ranging red-legged partridge (Alectoris rufa; 137 males and 130 females) were captured in 2009 (n = 101), 2010 (n = 130) and 2011 (n = 36). After biometric data and blood samples collection, animals were released on site. Blood samples were used for the preparation of blood smears. A total of 1.5% of smears presented parasites with a light blue finely granular cytoplasm in red blood cells. The parasites were identified, on the basis of their morphology (no molecular identification was possible), as being Haemoproteus sp. juvenile forms. The infestation prevalence was 0.8% in 2010 and 8.3% in 2011. No parasite was observed in 2009. The size of the infested erythrocytes was not altered (P > 0.05) and the nuclear displacement ratio was 0.8 ± 0.2. All the birds were apparently in good health status. To our knowledge this is the first description of Haemoproteus sp. in red-legged partridge, and more in general in birds belonging to Galliformes Order in Italy.

Nutritional value of meat lipid fraction from red-legged partridge (Alectoris rufa) obtained from wild and farmed specimens1.

The red-legged partridge (Alectoris rufa) is a feathered game species of great socioeconomic importance in its native range and also in the UK. The aim of this study was to present a detailed comparison of meat's lipid fraction obtained from wild and farm-raised specimens and simultaneously compare the breast and leg meat portions. Meat from wild specimens had a significant (P < 0.05) lower proportion of saturated fatty acid (less 5.1%) and presented better P/S and n-6/n-3 ratios, and atherogenicity index than farm-raised counterparts. The wild specimens presented significant (P < 0.001) higher contents of total vitamin E (8.8 vs. 2.2 µg/g of fresh meat), is for that reason less prone to lipid peroxidation than farm-raised specimens. Meat portions differed significantly (P < 0.05) on total lipid and total cholesterol contents and in all partial sums of fatty acids. The breast was leaner (0.86 vs. 1.47 g/100 g of meat), with lower total cholesterol (37.5 vs. 54.7 mg/100 g of meat), lower saturated fatty acid, monounsaturated fatty acid, polyunsaturated fatty acid, and n-6 polyunsaturated fatty acid (less 0.27, 0.28, 0.10, and 0.11 g/100 g of fresh meat, correspondingly). Regarding the fatty acid ratios and lipid quality indexes, breast meat presents better n-6/n-3 ratio and atherogenicity and thrombogenicity indexes.

Susceptibility and role as competent host of the red-legged partridge after infection with lineage 1 and 2 West Nile virus isolates of Mediterranean and Central European origin.

West Nile virus (WNV; genus Flavivirus; family Flaviviridae) is the aetiological agent of an emerging, mosquito-borne disease with great impact on human and animal health. Over the past 15 years, WNV has been responsible for large epidemics mainly in North America but also in Europe, where lineage 1 and more recently lineage 2 strains have caused an upsurge in the number of outbreaks with increased human infection and higher virulence for certain wild bird species. This study aimed to compare the course of infection of the lineage 1 WNV strains Israel/98 and Italy/08 and the lineage 2 strain Austria/08 in the red-legged partridge (Alectoris rufa), a gallinaceous bird indigenous to the Iberian Peninsula and widely distributed in Southern and Western Europe. After experimental inoculation, clinical and analytic parameters (viraemia, viral load, antibodies) were examined over a period of 15 days. All inoculated birds became viremic and showed clinical disease, with a morbidity rate of 100% and mortality rates between 22.2 and 55.5% depending on the virus strain. The red-legged partridge demonstrated to be a competent host for transmission of the three investigated WNV isolates with the highest competence index observed for the Italian strain. Likewise, this strain was the most pathogenic causing the highest viral loads in blood, organs, feathers and oral and cloacal secretions. These experimental results indicate that the red-legged partridge is highly susceptible to the infection with lineage 1 and 2 WNV strains and that this species may act as an amplifying host for both WNV lineages.

Experimental North American West Nile Virus Infection in the Red-legged Partridge (Alectoris rufa).

After the introduction of West Nile virus (WNV) into North America, bird mortalities associated with West Nile disease have dramatically increased in this continent and, to a lesser extent, in Europe. The different West Nile disease incidence in birds in these 2 continents demands an explanation, and experimental studies can provide important information. The authors inoculated thirteen 9-week-old red-legged partridges (Alectoris rufa) with 10(7)plaque-forming units of a WNV strain isolated in New York in 1999. The objective was to study the pathogenesis of the infection in a native Euro-Mediterranean bird species with a WNV strain known to be highly pathogenic for numerous native American bird species. Additionally, the authors evaluated the dynamics of inflammatory cell activation and recruitment into the brain. WNV was detected in tissues 3 days postinoculation (dpi), and the birds developed macroscopic and microscopic lesions. Two partridges succumbed to the disease. The most affected tissues were the heart, brain, and spinal cord. The main microscopic findings were the presence of mononuclear infiltrates in the heart and brain, gliosis, and degeneration and necrosis of cardiomyocytes and neurons. These lesions were aggravated in the birds that died or were euthanized 7 dpi or later. In the brain, there was an upregulation of microglial cells and astrocytes and an increase in the number of T cells, especially after 7 dpi. These results show that this WNV strain is of moderate virulence for the red-legged partridge and that WNV-infected red-legged partridges develop an immune cell response in the brain similar to that of mammals.

Are parent-reared red-legged partridges (Alectoris rufa) better candidates for re-establishment purposes?

Aiming to produce game birds suitable for re-establishment purposes, we studied the survival and behavior of 31 adult red-legged partridges reared by their natural parents in captivity (parent-reared) and 67 wild red-legs (35 adults and 32 subadults). Birds were radio-tracked and released in a game estate where management targeting small game species was conducted and shooting was not allowed. Survival of parent-reared partridges was shorter (mean 108 d) than wild adults (mean 160 d), though no significant differences were found. Parent-reared birds showed a longer escape reaction (mean 11.7 sec) than wild adults (mean 0.4 sec) and were mainly predated by terrestrial predators (52%), whereas the most important source of predation in wild red-legs was raptors (49%). The home range of parent-reared (mean 23 ha) was not significantly lower than wild adults (mean 27 ha). Nineteen percent of parent-reared and 31.4% of wild adults paired successfully, and we recorded 6 pairs in which one bird was wild and the other parent-reared and one pair in which both birds were parent-reared. A small proportion of red-legs started incubation (parent-reared 12.9%, wild 20%), and the proportion completing incubation was similar among wild and parent-reared birds. Overall, parent-reared red-legs showed similar survival and behavior compared to wild red-legs and better than intensively reared, so parent-reared red-legs should be chosen for the re-establishment of wild populations.

Effects of storage temperature and length of the storage period on hatchability and performance of red-legged partridge (Alectoris rufa) eggs.

The aim of the present study was to investigate, in red-legged partridge (Alectoris rufa) eggs, the effects of 7- and 42-d storage periods with different storage temperatures (15, 12, and 9°C) on egg weight loss, hatchability, chick weight at hatch, incubation length, and development stage at embryonic mortality. A total of 420 red-legged partridge eggs were arranged in a 2 × 3 factorial design with 2 levels of storage length and 3 levels of storage temperature, resulting in 6 treatments consisting of 10 replications of 7 eggs each. We found that the storage length significantly reduced hatchability of the fertile eggs (P = 0.001), increasing late embryonic mortality (P = 0.001). Storage temperature did not influence on the embryonic mortality at any stage (P > 0.05). Egg weight loss during storage increased with the storage length (P < 0.001), storage temperature (P < 0.001), and their interaction (P < 0.001). Incubation length increased with the storage length (P < 0.001); however, it was not influenced by the storage temperature (P > 0.05). Nevertheless, incubation period decreased with the storage temperature for 7-d storage, and increased with the storage temperature for 42-d storage (P = 0.005). It can be concluded that in this study red-legged partridge eggs stored well with little deterioration up to 42 d at 9 and 12°C and 80% RH, in contrast to the lesser durability of eggs described in the literature for other poultry species. In case of 7-d storage periods, hatchability of A. rufa fertile eggs is higher when they are stored at 15°C. These findings are useful to address specific demands of game farms that require fertile eggs for hatching whose shelf-life should be long enough to maintain hatchability until further incubation. And, due to the marked reproductive seasonality of red-legged partridge, long-term storage of hatching eggs could permit the distribution of batches of chicks throughout the year.

Oculopathologic findings in flavivirus-infected gallinaceous birds.

Using eye samples of nine 9-week-old experimentally West Nile virus (WNV)-infected red-legged partridges (Alectoris rufa), time course of lesions and WNV antigen appearance in ocular structures were examined. In addition, eye samples of 6 red-legged partridges and 3 common pheasants (Phasianus colchicus) naturally infected with Bagaza virus (BAGV) were used to study lesions and flavivirus antigen distribution in relation to apparent blindness in the former. The rapid onset of microscopic lesions and early presence of viral antigen in the eye of experimentally WNV-infected partridges, prior to the central nervous system involvement, suggested hematogenous spread of the virus into the eye. BAGV-infected partridges had a more pronunced inflammatory reaction and more widespread flavivirus antigen distribution in the retina compared with pheasants and experimentally fatally WNV-infected partridges. Our results suggest that flavivirus replication and development of lesions in ocular structures of gallinaceous game birds vary with the specific virus and host species involved.

Physical characteristics of the eggs of red-legged partridge (Alectoris rufa) reared in captivity / Características físicas dos ovos da perdiz vermelha (Alectoris rufa) criada em cativeiro

O objetivo do presente estudo foi avaliar os efeitos da idade das fêmeas (um, dois e três anos) e do mês de postura (março, abril e maio) sobre as características físicas dos ovos da perdiz vermelha (Alectoris rufa) criada em cativeiro. O peso (W), o comprimento máximo (L) e a largura máxima (B) de 2878 ovos foram determinados diretamente, enquanto o índice de forma (B/L), o volume (V) e a superfície (S) foram calculados com base nos parâmetros determinados diretamente. A análise mostrou diferenças significativas (P<0,01) no peso dos ovos entre as diferentes idades e entre meses de postura, com menor peso nas fêmeas mais jovens. Observaram-se diferenças significativas (P<0,01) no comprimento do ovo entre as classes de idade, mas não entre os meses de postura (P>0,05). Observaram-se diferenças significativas (P<0,01) na largura máxima e no índice de forma do ovo entre as diferentes classes de idades, com valores mais elevados nas fêmeas mais velhas e no período de postura mais tardio. O volume dos ovos estimados por meio de V1= 0,51LB2e V2=0,913W foi afetado significativamente (P<0,01) pela idade e pelo mês de postura, bem como as áreas, S1=4.835W0,662, S2=4,951V10,666e S3=4,951V20,666, as quais apresentaram os mesmos efeitos.