Identification of Sarcocystis spp. in Slaughtered Sheep from Spain and Evaluation of Bradyzoite Viability after Freezing.

Sarcocystis spp. are complex apicomplexan parasites that cause a substantial economic impact on livestock used for meat production. These parasites are present worldwide. Our study aimed to identify Sarcocystis species affecting sheep meat in southern-central Spain and to evaluate the effectiveness of freezing for parasite inactivation. A total of 210 condemned samples of sheep meat were thoroughly assessed grossly and microscopically; the presence of macro- and microcysts was confirmed. The samples were then frozen at -20 °C for various time intervals (24, 48, 72, 96, 120, and 144 h) and compared with untreated samples. Bradyzoites were isolated through pepsin digestion for subsequent molecular analysis and viability assessment, employing trypan blue and double fluorescence staining techniques. Our measurements confirmed the presence of S. tenella, S. gigantea, and S. medusiformis in Spanish domestic sheep. Freezing for 96 to 144 h resulted in a significant reduction in parasite viability, with a robust correlation observed between the two staining methods. Both stains effectively measured the viability of Sarcocystis, thereby promising future advances in meat safety.

A non-replicative antibiotic resistance-free DNA vaccine delivered by the intranasal route protects against canine leishmaniasis.

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis (ZVL). The disease is endemic in Central and South America, Central and South East Asia, and the Mediterranean basin. Dogs are the main reservoir, with an estimated prevalence of approximately 2.5 million dogs in Southern Europe. Current treatments cause side effects, disease recurrence, and drug resistance. Therefore, the development of vaccines against canine leishmaniasis is necessary. We have generated a DNA vaccine based on the non-replicative antibiotic resistance marker-free plasmid vector pPAL that contains the encoding gene for the L. infantum activated protein kinase C receptor analog (LACK). Homologous pPAL-LACK prime-boost intranasal administration confers efficacious protection in Beagle dogs with a reduction of clinical signs and a statistically significant reduction of the parasite burden in the bone marrow of more than 90% of dogs after experimental infection with highly infective promastigotes. This DNA vaccine elicits a robust cellular immune response skewed towards the Th1 profile.

Toxoplasma gondii in meat of adult sheep in Spain.

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, an intracellular parasite that presents a worldwide risk. Humans can become infected by ingesting meat infected with T. gondii, and the consumption of infected sheep and goat meat is a significant public health issue. Antibodies against T. gondii have been found in sheep in Spain, indicating the presence of the parasite in the country. However, no previous studies have assessed the presence of T. gondii in sheep meat in Spain. In view of the significance of the transmission of T. gondii through meat consumption and given the lack of previous studies in Spain, we carried out an investigation to evaluate the presence of T. gondii in adult sheep meat (mutton). A total of 216 muscle samples were analyzed by digestion, and a real-time PCR assay was used to determine the presence of T. gondii DNA. A total of 24.5% of the samples were found to be parasitized, indicating that the consumption of sheep meat can present an important risk for human health.

Atypical Lesions in Canine Leishmaniosis: Description of New Cases.

Pathological characteristics are well described in canine leishmaniosis (CanL). However, atypical lesions that can be confused with other pathologies or trigger unusual clinical signs are sporadically reported. Atypical lesions were observed during routine postmortem examination in three Leishmania-infected dogs and samples were taken for histopathological and immunohistochemical studies. Clinical signs, biochemical parameters, level of antibodies, and parasite detection by PCR were also evaluated. Atypical lesions were found in the peritoneal cavity, liver, and spleen. Splenomegalia and hepatomegalia were observed in all dogs. In addition, multifocal dark to white nodules of variable size were observed in the peritoneal cavity, liver, and spleen of one dog and in the spleen of the other two dogs. One dog presented diffuse irregular whitish lines with a threadlike appearance and another an intense fibrotic depression in the intermediate lobe. Microscopically, an intense granulomatous inflammation with abundant macrophages, a variable number of lymphocytes, and a low to moderate number of parasites was observed. This study represents the first description of granulomatous peritonitis associated with Leishmania in dogs. It also shows atypical macroscopic expression of hepatitis in CanL. In the absence of an adequate clinical history and laboratory analyses, certain lesions observed in CanL could admit alternative diagnoses.

Characterization of lesions in the temporal muscle and the male reproductive system (epididymis and testicle) of dogs experimentally infected with Leishmania infantum with different clinical stages.

Leishmaniosis is a zoonotic disease with a very complex pathogenesis modulated by the interaction between the parasite, the vector and the host. Although the pathological characteristics have been extensively studied in the typically affected organs, some locations such as muscles and reproductive organs have been less studied. The objective of this study was to evaluate the presence of lesions in the temporal muscle and the male reproductive organs (testicle and epididymis) and correlate their characteristics with the presence of the parasite and with the clinical status of the dogs. The temporal muscle was studied in 25 infected beagle dogs (nine females and 16 males) and five uninfected control dogs (two females and three males) and the testicle and epididymis in the 19 males. Dogs were euthanized one year after infection and clinical signs, anti-Leishmania serum antibodies, and lymph node parasite load were assessed. Muscular and reproductive lesions were characterized by H&E and immunohistochemistry (IHC). The presence of the parasite in the lesions was evaluated using IHC and molecular techniques. Myositis was observed in 72% (18/25) of the dogs and was characterized by lymphoplasmacytic or histiocytic lesions. Mild and severe lesions were detected, the latter being statistically associated with the presence of the parasite and with the clinical status of the dogs. Orchitis was observed in 50% (8/16) of the dogs and was mainly mild and lymphoplasmacytic. No statistical relationship was found between testicular lesions and the presence of the parasite or the clinical status. Epididymitis was observed in 87.5% (14/16) of the dogs, and the lesions were often infiltrated by numerous histiocytes and neutrophils. Epididymal lesions were statistically associated with the clinical status of the dogs and with the presence of the parasite in the lesions. IgG and IgM immunoglobulins were found in all lesions, suggesting a local immune response with reactivation of the infection. Leishmania was more frequently detected in severe and histiocytic lesions, although some lesions had no detectable parasites. These results have shown that lesions in the temporal muscle, epididymis, and testicles are common in dogs infected by Leishmania infantum and that dogs may show a different response to infection. This response is characterized by varying degrees of cellular and immune responses associated with a variable presence of the parasite.

Comparative Study of Real-Time PCR (TaqMan Probe and Sybr Green), Serological Techniques (ELISA, IFA and DAT) and Clinical Signs Evaluation, for the Diagnosis of Canine Leishmaniasis in Experimentally Infected Dogs.

Canine leishmaniasis (CanL) diagnosis is not fully resolved. Currently, two specific methodologies are in continuous development, the detection of the parasite DNA or RNA in target organs and the detection of specific antibodies against Leishmania sp. For a correct diagnosis, it has been shown that the joint use of this type of test is necessary. In this work, a Sybr Green and a TaqMan Probe based on real time PCRs (qPCR) was performed for the detection of Leishmania sp. in order to correlate the results with clinicopathological and serological evaluations (IFA, ELISA and DAT) to propose an optimal biological sample to be used to detect the parasite in both early and late stages of the infection. A total of four samples were processed: conjunctival swabs, popliteal lymph node aspirates, bone marrow aspirates, and peripheral blood from experimentally infected dogs belonging to a larger study. Our results indicated that a single non-invasive sample (conjunctival swab) and the application of both types of qPCR would be reliable for determining Leishmania infection as well as the disease stage in dogs, thus avoiding bone marrow, lymph node aspirate or blood samples collection.

Geochemical fingerprint of agricultural liming as a regular management practice in Modern-period Basque farming.

The Modern period in Europe is marked by the introduction of deep agricultural changes. In the Basque Country (northern Spain), the implantation of an intensive crop rotation was made possible by the expansion of agricultural liming, although the extent and implications of this practice have not been previously explored in depth. The present paper proposes a multidisciplinary approach to this question, based on the combined analysis of archival sources, toponymy, visual prospection focused on the presence of limekilns, and agricultural soil coring in four local contexts of the Atlantic Basque Country. The results show, for the first time, evidence of concurrent and widespread liming in this territory at the edge of the 18th century, with strong implications for the model of agricultural management in the communities involved. The spreading of mineral fertilisation reflects an intensification in the forms of agricultural management, in the framework of a new relationship between land and labour that emerged after the introduction of American crops. Continuous liming for more than 200 years exerted a deep impact in the analysed soils, with interesting socio-economic and ecological implications that are representative of the potential short-term effects that changing relationship between humans and their socio-ecological environment may produce in agricultural soils.

Prevalence and risk factors of gastrointestinal nematode infestation of horses, donkeys and mules in tropical, dry and temperate regions in Mexico.

The aims of this study were to determine the prevalence of gastrointestinal nematodes (GIN) in horses, donkeys and mules and its associations with age, sex and climatic factors (derived from satellite data) to identify the potential risk factors of different climate regions in four Mexican states. From May 2017 to April 2018, a total of 560 farm owners or managers answered the questionnaire, and the data were used to establish three Köppen climate classes (tropical, dry, temperate). The overall prevalence of GIN parasites in equines was 77.9% (436 out of 560). The highest percentage of GIN was detected in mules (87.5%). In addition, the highest prevalence among the climate regions was found in the temperate climate (79.9%). The identified nematodes were strongyle type (77.9%), Parascaris equorum (5.7%), Strongyloides (0.7%) and Oxyuris equi (2.9%). The highest mean value of strongylid eggs per g faeces was found in the dry climate (632.6 ± 96.8), followed by that in the tropical climate (518.3 ± 49.7) and temperate climate (383.8 ± 30.2); however, a similar prevalence was observed between different climate regions. Age and sex were identified as risk factors for high shedding of strongyle eggs: the odds ratios for higher shedding intensities were 3.858 for geldings compared to mares, 2.602 for 6-10-year-old equines and 3.597 for ≤16-year-olds compared to young equines (≤5 years old).

Neutrophil Gelatinase-Associated Lipocalin (NGAL) Is Related with the Proteinuria Degree and the Microscopic Kidney Findings in Leishmania-Infected Dogs.

Early diagnosis of renal damage in Leishmania infected dogs may allow appropriate treatments and prevent some deaths. This study investigates neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker of kidney disease in dogs experimentally infected with Leishmania infantum. Serum, urine, and kidney samples were collected from 30 infected beagle dogs and six uninfected control dogs. Based on proteinuria and azotemia values, dogs were initially classified. NGAL was measured in urine and serum samples. Then, the urinary NGAL to creatinine ratio (uNGAL/C) was calculated. Kidney samples were taken for histopathological studies, and the dogs were classified according to the severity of glomerular and tubulointerstitial lesions. In Leishmania-infected dogs, the uNGAL/C was significantly higher in proteinuric non-azotemic dogs compared with non-proteinuric non-azotemic dogs (p = 0.038). Serum NGAL (sNGAL) concentration did not differ between groups. Microscopic studies revealed several degrees of glomerulonephritis and slight focal lymphoplasmacytic interstitial nephritis in 89% and 55% of infected dogs, respectively. Urinary protein to creatinine ratio (UPC) and uNGAL/C were significantly higher in dogs with affected glomeruli compared to infected dogs without renal lesions (p = 0.045 and p = 0.043, respectively). The results show that uNGAL/C correlates with proteinuria and the presence of moderate glomerular lesions in non-azotemic dogs experimentally infected with L. infantum.

Pathological findings in genital organs of bulls naturally infected with Besnoitia besnoiti.

Bulls chronically affected by bovine besnoitiosis can suffer from sterility. There is limited information about the distribution of Besnoitia cysts and their associated lesions within the male genital organs. This work describes the gross and histological abnormalities in the genital organs of 6 bulls chronically infected with Besnoitia besnoiti, including both clinically (n = 4) and subclinically (n = 2) affected cases. Parasitic cysts were observed in the genital organs of all the clinically affected bulls. The tissue cysts were most commonly found within the pampiniform plexus (4/4), where they were often seen within venous vascular walls and associated with vasculitis, followed by epididymis (3/4), tunica albuginea (2/4), and penis (1/4). In decreasing order of their frequency, observed abnormalities included seminiferous tubule degeneration, testicular fibrosis, testicular necrosis, lack of/or diminished numbers of spermatozoa, testicular atrophy, and Leydig cell hyperplasia. Only one of the subclinically infected bulls had few Besnoitia cysts within the pampinoform plexus, which was associated to small areas of necrosis and mineralization in the ipsilateral testicle. Results indicate that Besnoitia cysts and genital abnormalities are frequent in bulls chronically affected by bovine besnoitiosis, while they are mild and scarce in subclinically affected ones. Moreover, present data show that Besnotia-associated testicular lesions can occur without the presence of cysts within the testicular parenchyma. B. besnoiti cysts seem to have a tropism for the vascular structures of the spermatic chord, which may cause testicular abnormalities via vascular damage, reduced blood flow, and/or impaired thermoregulation and subsequently lead to the observed testicular lesions.

First database of the spatial distribution of Eimeria species of cattle, sheep and goats in Mexico.

Ruminant coccidiosis is a gastrointestinal disease caused by parasites of the genus Eimeria. Environmental and climatic factors are relevant for the development, survival, and transmission of coccidiosis because Eimeria oocysts are able to survive in the environment for several weeks or months in favorable conditions of moderate heat and moisture. The aim of the present study was to georeference, for the first time, the locations of Eimeria occurrences in Mexico from 1961 to 2018. A dataset was created for 3414 reports of Eimeria occurrences in cattle, sheep, and goats in Mexico. Twelve species of Eimeria that infect cattle were recorded, 11 Eimeria species of sheep are present in Mexico, and eight species of goats are geographically distributed in the country. In the current findings, it has been observed that Eimeria colonizes mainly the temperate semihumid, temperate humid, warm humid, and warm semihumid areas during spring and summer in animals younger than 1 year of age. Macroenvironmental variables like temperature and rainfall influence the prevalence of Eimeria in cattle, sheep, and goats, and for some species, the rearing system, facility type, farm size, and altitude affect the occurrence of this parasite. Results may support future studies aimed at reducing the disease prevalence of the parasite in endemic regions of Mexico. The use of recorded cases and climate variables yields a more comprehensive perspective of the epidemiology of eimeriosis, which would be difficult to infer from laboratory studies alone.

On the origin of rural landscapes: Looking for physico-chemical fingerprints of historical agricultural practice in the Atlantic Basque Country (N Spain).

Evolution and change in agricultural practice is a major factor in the codification of social relations and represents one of the main resources employed by human societies to establish a durable relationship with their environment. Using a multi-proxy integrated approach, this paper seeks to decipher the long-term dynamics that have shaped agricultural landscapes in the Basque Country (N Spain). Social and economic indicators (archival records, toponymy and oral sources) are used along with geological core sampling (geochemistry, magnetic, palynological and carpological analyses) to reconstruct a diachronic sequence of human settlement and agricultural management in the village of Aizarna over the last ~1500 years. The oldest records obtained refer to non-agricultural human activities dating back to the Roman period. Later on, traces of agricultural landscape-transformation can be divided into four main phases: 1) the onset of terraced agriculture, defined by the clearance and terracing of previous forested areas during the Early Middle Ages; 2) a Late Medieval reorganisation, with new terraces being (re)constructed close to dispersed farmsteads, linked to the emergence of the modern rural landscape; 3) a new model of intensive polyculture developed during the Modern period as a consequence of the introduction of new crops of American origin; and 4) the mechanisation and commercialisation of the agricultural production over the 20th century. These results provide a valuable pathway for the investigation of currently inhabited rural contexts, and offer, for the first time in this region, an overview on long-term landscape construction in the Atlantic areas of the Basque Country.

A randomized, blinded, controlled trial to assess sand fly mortality of fluralaner administered orally in dogs.

BACKGROUND: Leishmania infantum is the parasite responsible for the disease in humans known as zoonotic visceral leishmaniasis (ZVL). Dogs are considered the main domestic reservoir of ZVL and sand flies are the proven vectors. The use of systemic insecticides in dogs has been studied as an alternative strategy to control ZVL in endemic areas. One systemic insecticide in dogs, fluralaner, has a proven anti-sand fly effect in membrane-fed studies. However, the efficacy and duration on sand flies directly feeding from dogs treated with fluralaner remains unknown. METHODS: Direct feeding bioassays were performed on 10 beagle dogs that had been randomly assigned to two groups: one with five dogs orally treated with Bravecto® (fluralaner) and other five as a control. About 30 females of Phlebotomus papatasi were allowed to directly feed from dogs at seven days before the administration of the treatment and Days 3, 17, 31, 45 and 73 post-treatment. Sand fly mortality after feeding was observed every 24 h for 5 days. The Kaplan-Meyer method, Henderson-Tilton formula and a negative binomial mixed model were used to respectively calculate: (i) mortality and its 95% confidence interval (CI); (ii) efficacy of the insecticide at killing sand flies in 24 h; and (iii) differences in the risk of sand fly death at 24 h after feeding. RESULTS: Control sand fly mortality 24 h after feeding was always ≤ 20% and mortality in the fluralaner group ranged from 2% (95% CI: 0-4%) 7 days before treatment to 100% at 3 days post-treatment. Fluralaner efficacy was 100, 93, 94 and 75% at Days 3, 17, 31 and 45, respectively (P < 0.0001). The increase in the risk of sand fly death was 32.9 (95% CI: 4-263), 76 (95% CI: 8-705), 95.8 (95% CI: 9-1029) and 10.6 times (95% CI: 1.43-79) on Days 3, 17, 31 and 45, respectively CONCLUSIONS: The efficacy of fluralaner, orally administered to dogs, against sand-flies was above 90% for 31 days. Fluralaner administered to dogs should be further evaluated as a control strategy in ZVL endemic areas.

Development of the first georeferenced map of Rhipicephalus (Boophilus) spp. in Mexico from 1970 to date and prediction of its spatial distribution.

The tick genus Ripicephalus (Boophilus), particularly R. microplus, is one of the most important ectoparasites that affects livestock health and considered an epidemiological risk because it causes significant economic losses due, mainly, to restrictions in the export of infested animals to several countries. Its spatial distribution has been tied to environmental factors, mainly warm temperatures and high relative humidity. In this work, we integrated a dataset consisting of 5843 records of Rhipicephalus spp., in Mexico covering close to 50 years to know which environmental variables mostly influence this ticks' distribution. Occurrences were georeferenced using the software DIVA-GIS and the potential current distribution was modelled using the maximum entropy method (Maxent). The algorithm generated a map of high predictive capability (Area under the curve = 0.942), providing the various contribution and permutation importance of the tested variables. Precipitation seasonality, particularly in March, and isothermality were found to be the most significant climate variables in determining the probability of spatial distribution of Rhipicephalus spp. in Mexico (15.7%, 36.0% and 11.1%, respectively). Our findings demonstrate that Rhipicephalus has colonized Mexico widely, including areas characterized by different types of climate. We conclude that the Maxent distribution model using Rhipicephalus records and a set of environmental variables can predict the extent of the tick range in this country, information that should support the development of integrated control strategies.

Variations in seroprevalences of canine leishmaniasis: Could it be a consequence of the population structure?

Canine leishmaniasis is a parasitic disease caused by Leishmania infantum and is transmitted by Phlebotominae vectors. Despite numerous publications on the subject, some essential aspects of the epidemiology are not yet sufficiently clear. We proposed a stochastic model with the aim of identifying some important gaps in the current knowledge of leishmaniasis, such as the frequency of vector infection or a dog's life expectancy depending on their purpose and their health status. We only found that the purpose was a significant factor. Furthermore, we detected relationships among age, gender and habitat with the dogs' purposes that can affect the calculation of the overall seroprevalence of the analysed sample. The development of this model will allow us to discard potential confounding factors as gender, age, purpose or habitat.

Proteolytic activity of extracellular products from Arthrobotrys musiformis and their effect in vitro against Haemonchus contortus infective larvae.

Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolytic activity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolytic activity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolytic activity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of A musiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs.

Prevalence of Besnoitia besnoiti infection in beef cattle from the Spanish Pyrenees.

Reports of recent outbreaks of bovine besnoitiosis in Europe have emphasized the need for prevalence studies to determine the importance of the disease. A previous cross-sectional study carried out in a northern province of Spain showed that seropositive cattle were beef breeds primarily located in regions where the disease is endemic (such as in the Pyrenees). The present study focused on this population. Sixty-three beef herds were selected and at least 50% of the animals in each herd were sampled (n = 3318). The herd, intra-herd and animal prevalence rates were calculated. All breeding bulls located in the same region were also sampled (n = 587), and the animal prevalence calculated. Sera were tested by ELISA. The herd prevalence rate of bovine besnoitiosis was 87.3%, and both sexes were similarly affected (approximately 50%). The results indicate that the disease is highly widespread in the Pyrenees, and that serological examination of cattle should be recommended when trading beef cattle.

How to increase the population of a Phlebotomus perniciosus (Diptera: Psychodidae) colony: a new method.

The sandfly Phlebotomus perniciosus is the most widespread vector of Leishmania infantum in Spain. Laboratory colonisation represents the most feasible source of information on the biology of these insects, but in conducting any study, the density of individuals in the colony may drop to such an extent that it is sometimes difficult to recover the initial population levels. A new technique was tested for the recovery of sandfly eggs in three different colonies; the recovery rate was studied by comparing the standard method of mass rearing with this new method of colony management. The results demonstrate a mean increase of 18.4% in adult production, a growth in colony productivity that justifies the inclusion of this process in the routine maintenance of any colony of sandflies.

How to increase the population of a Phlebotomus perniciosus (Diptera: Psychodidae) colony: a new method

The sandfly Phlebotomus perniciosus is the most widespread vector of Leishmania infantum in Spain. Laboratory colonisation represents the most feasible source of information on the biology of these insects, but in conducting any study, the density of individuals in the colony may drop to such an extent that it is sometimes difficult to recover the initial population levels. A new technique was tested for the recovery of sandfly eggs in three different colonies; the recovery rate was studied by comparing the standard method of mass rearing with this new method of colony management. The results demonstrate a mean increase of 18.4 percent in adult production, a growth in colony productivity that justifies the inclusion of this process in the routine maintenance of any colony of sandflies.