Evaluación del cultivo fecal de pools y de la qPCR en muestras individuales y su aplicación en el diagnóstico de la paratuberculosis en vacuno de leche

Los objetivos principales de este trabajo fueron establecer un plan de control de la paratuberculosis de tres años de duración basado en el cultivo fecal de pools de todos los animales mayores de dos años presentes en las explotaciones incluidas, así como comparar la clasificación sanitaria obtenida mediante este método con respecto a los resultados serológicos obtenidos el primer año de estudio. También se comparó la utilidad del cultivo fecal de pools con el cultivo fecal individual para la monitorización del estatus sanitario de los rebaños, y se estudiaron dos frecuencias de muestreo para la realización de los cultivos fecales de pools (semestral/anual). Otro de los objetivos planteados fue la posible aplicación de este método para evaluar la reducción de la prevalencia individual tras la implantación en granja de medidas de bioseguridad y manejo higiénico. Por último, también se comparó la validez de una técnica de qPCR basada en la diana F57 en relación a los resultados obtenidos mediante cultivo fecal individual. La parte experimental de este trabajo se dividió en tres estudios, que se realizaron entre los años 2009-2012 en 36 rebaños de vacuno lechero de la provincia de Lugo. Los resultados del primer estudio muestran una escasa concordancia entre los resultados de serología y cultivo fecal de pools para la clasificación sanitaria de las granjas, fundamentalmente debido a granjas seropositivas pero con cultivo negativo. Al estratificar los resultados en función de la edad de los animales, se pudo observar que el mayor grado de concordancia se producía en el grupo de edad de 2 años, disminuyendo progresivamente a medida que aumentaba la edad de los animales. Este hecho hace sospechar de un problema de especificidad de las técnicas serológicas ya que, a medida que aumenta la edad de los animales, aumenta la probabilidad de contacto con otras micobacterias y por tanto la probabilidad de obtener falsos resultados positivos. El segundo estudio permitió observar que el porcentaje de explotaciones positivas detectadas mediante cultivo fecal de pools aumentaba a cada año de muestreo, poniendo de manifiesto la utilidad de este sistema para la monitorización de los rebaños y la necesidad de que se realice de manera continuada. Mediante el cálculo de la prevalencia individual a partir de los resultados de los cultivos de pools fecales se comprobó que este método es una herramienta válida para evaluar la prevalencia individual y, por tanto, la efectividad de los protocolos de bioseguridad y manejo implantados. En cuanto a la comparación con el cultivo fecal individual y el aumento de la frecuencia de análisis, ninguna de estas variaciones mejoró los resultados obtenidos mediante cultivo fecal de pools para la clasificación sanitaria de las explotaciones. Además, se identificaron diferentes factores de riesgo mediante la clasificación obtenida en el último año, lo que ilustra también la necesidad de que los estudios de factores de riesgo se realicen en granjas que presenten un control de la enfermedad como mínimo de tres años. Los resultados del tercer estudio muestran que la técnica de qPCR basada en la diana F57 es adecuada para sustituir al cultivo microbiológico individual, reduciendo en gran medida el tiempo de diagnóstico, y sugiriendo además que esta técnica de PCR podría detectar la eliminación fecal más precozmente que las técnicas microbiológicas convencionales en aquellos casos en los que la eliminación fecal es muy baja

Efficacy of two commercial ready-to-use PCV2 and mycoplasma hyopneumoniae vaccines under field conditions

Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae are economically important pathogens in swine farms. Vaccination is the main preventive measure for both infections. In order to test two ready-to-use bivalent vaccines, 646 piglets from a herd actively infected with both pathogens were stratified according to the sow parity number and randomly assigned to three groups: A and B were vaccinated with two different vaccines, respectively, while C remained as the unvaccinated control. Vaccine efficacy was assessed based on the weight, average daily weight gai (ADWG), degree of lung lesions, presence of PCV2 viremia by qPCR and presence of PCV2 and M. hyopneumoniae antibody levels by ELISA. Our data revealed that the sow parity did not influence the vaccine outcomes. Good results for most of the analyzed parameters were observed in both vaccinated groups. ADGW and final weight were higher and lung lesions were less evident in both vaccinated groups than in the control one, but only Group A showed a significant improvement. PCV2 viremia was not detected in Group A, but it did appear in Group B coinciding with its peak in Group C. Finally, both the PCV2 and M. hyopneumoniae serological patterns differed depending on the employed vaccineS

Validation of a simple method for the interpretation of uterine cytology in cows

One of the main drawbacks of using endometrial cytology in cows is the time required for sample collection and interpretation. It is recommended to count a large number of polymorphonuclear neutrophils (PMN) and to calculate their overall percentage. However, since counting a large number of cells is a laborious method, it would be preferable to simplify the analysis by counting the number of PMN in few microscopic fields. Therefore, the aim of this study was to assess whether a simple test, based on calculating the average number of PMN in 10 fields at 1000×, could be a reliable technique for the diagnosis of endometritis. Two hundred and sixty endometrial samples were taken from Holstein cows at different postpartum stages using an adapted cytobrush. Smears obtained were air-dried for fixing and stained with a Romanowsky-type procedure. To evaluate the counting method, the percentage of PMN in 150 cells was calculated as well as the average number of PMN in 10 fields at 1000×. Receiver operating characteristic (ROC) curves was constructed to evaluate both methods, the percentage of PMN (used as reference) and the average number of PMN. It was observed that the area under the curve is (regardless of cut-off used) higher than 0.99 and the correspondence between both methods were 1.58 PMN/field for the cut-off value of 15% and 2.40 PMN/field for the cut-off value of 20%. These results show that this simple method could be used to determine the percentage of PMN in endometrial cytological samples and to diagnose endometritis in cowsSupported by the Xunta de Galicia (Galician Plan for Research and Technological Development; Grant No. PGIDIT07MRU002E) and the Friesian Federation of Galician, A Coruna, SpainS

Environmental distribution of Porcine Circovirus Type 2 (PCV2) in swine herds with natural infection

Porcine circovirus type 2 (PCV2) is the aetiological agent of PCV2-Systemic Disease (PCV2-SD) and PCV2-Subclinical Infection (PCV2-SI). PCV2 is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance. The aim of this study was to identify, under field conditions, the possible critical points in the environment of non-vaccinated farrow-to-weaning swine farms where PCV2 could accumulate and persist. For that, environmental samples from five swine farms with PCV2-SD or PCV2-SI were taken and analysed by qPCR, including different farm areas, farm personnel and management implements. PCV2 DNA was detected in the environment of all farms (42.9% of positive samples). Overall, the PCV2-SD herd seemed to present more positive samples and higher viral loads than the PCV2-SI herds. At individual farm level, weaning areas appeared to be the most contaminated facilities. In addition, PCV2 was found at high levels in most samples from farm workers, especially work boots, suggesting that they may play a role in within-farm transmission. In addition, PCV2 was detected in areas without animals the like warehouses, offices and farm perimeter. Therefore, this study is helpful to improve measures to reduce within-farm PCV2 dissemination.This work has been funded by the European PCV2-Award 2016 sponsored by Boehringer Ingelheim Animal Health, GermanyS

A case report of fatal feline babesiosis caused by Babesia canis in north western Spain

In Europe, Babesia infections in cats are sporadic and only partial knowledge is currently available since the number of described cases including both the clinical presentation and the molecular identification of the Babesia species involved is limited. In the present case report, the clinical signs, the epidemiological data and the molecular results suggest that this is the first reported fatal case of feline babesiosis caused by Babesia canisThis research was funded by the Program for consolidating and structuring competitive research groups (ED431C 2019/04, Xunta de Galicia, Spain). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS

Pedicle myiasis by Lucilia caesar (Diptera, Calliphoridae): an emerging disease in roe deer from north-western Spain

A total of 35 male roe deer (Capreolus capreolus) from Lugo province (north-western Spain) were examined for pedicle myiasis between 2020 and 2022. All these animals had died by different causes and were examined or preserved just after their death to avoid post-mortem infestations. After external and internal head inspection, five animals were diagnosed with severe myiasis at the basis of the antlers (14.29%; 95% confidence interval = 5.38–31.04). All the affected bucks presented a cutaneous wound (1.5–15 cm in diameter) around the pedicles with extensive tissue destruction, exposition of frontal and parietal bones, and massive infestation by dipteran larvae (2–12 mm long). Four of five roe deer showed whitish egg clusters adhered to the basis of the antlers. Moreover, in one animal, six larvae had penetrated the cranial cavity, and in two of them larvae were also found in the nasopharyngeal cavity. All the cases were recorded in summer (May–August). Morphological identification and subsequent molecular confirmation revealed that all animals were infested by different larval stages of Lucilia caesar (Diptera: Calliphoridae). This study represents the first report of pedicle myiasis in Spain. Since this disease is reported sporadically, the detection of five cases in a short period of time suggests an increase in the incidence of this myiasisS

The Goat as a Risk Factor for Parasitic Infections in Ovine Flocks

Small ruminants in northwestern Spain are frequently managed in mixed flocks. Sheep–goat joint management has not been considered a risk factor for parasite infection, so the main objective of this study was to establish if mixed management with goats supposes a risk factor for parasitic infections in ovine flocks. Two thousand and ninety-three sheep were sampled from 74 commercial meat ovine flocks for diagnostic of the main parasites. Goat contact was a risk factor for sheep to be infected by protostrongylids, Dictyocaulus filaria, gastrointestinal nematodes and Eimeria spp. In contrast, Moniezia, Fasciola hepatica, Dicrocoelium dendriticum and Paramphistomidae prevalences were not influenced. Sheep–goat mixed management can be considered as a risk factor, since goats would act as a source of pasture contamination for interspecific parasites (protostrongylids, Dictyocaulus filaria and gastrointestinal nematodes). In relation to host-specific parasites, such as Eimeria spp., goats cannot be considered as a source for sheep, but competition for food and spaces between both small ungulates can suppose a reduced grazing area to sheep, provoking high environmental contamination and stress that facilitate their infection. Future epidemiological studies for parasitic infections in small ruminants should consider sheep–goat mixed management as a possible risk factor to be included in multivariate analysesThis work was supported by a program for consolidating and structuring competitive research groups (ED431D 2019/04, Xunta de Galicia, Spain)S

Influence of subclinical endometritis on the reproductive performance of dairy cows

The aim of this study was to evaluate the influence of subclinical endometritis (SE) on the reproductive performance of dairy cows. Ninety-four dairy cows of parity 1 to 8, distributed in 25 herds, were examined once between 30 and 45 days in milk using transrectal palpation, vaginoscopy and ultrasonography. A cytological sample of the endometrium was taken only from cows with an apparent healthy uterus (n=65). Serum glucose, total cholesterol, triglycerides, non-esterified fatty acids, β-hydroxybutyrate, total proteins, albumin, urea and hepatic enzymes were analyzed. Reproductive indexes were recorded during the next 11 months. Endometrial cytology was considered indicative of SE if percentage of polymorphonuclear neutrophils was superior to 5% of all cells present in the smear, except erythrocytes. Results indicated that 14.9% of the cows sampled for uterine cytology had SE, and that healthy cows become pregnant significantly before than those with SE (hazard ratio=2.35; 95% confidece interval: 1.05-5.3). From all the metabolic and productive variables analyzed, only triglycerides affected negatively to reproduction; serum albumin concentration, body condition score and milk production had positive effects on the reproductive performance. In conclusion, our results indicate that SE has a negative impact on reproductive performance and uterine cytology is necessary to diagnose it since almost 15% of the affected animals were not detected by other diagnosis methodsXunta de Galicia (Programa Sectorial de Medio Rural, Proyecto Ref. PGIDIT07MRU002E) and FEFRIGA, Santiago de Compostela, SpainS

Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain

Metastrongyloid and trichuroid cardiorespiratory nematodes of dogs and cats are considered emergent in several European countries, and an increase in the number and extent of endemic foci has been described. Since data on their prevalence are limited in this continent, faecal samples from new arrivals (365 dogs and 65 cats) at two animal shelters in North-western Spain were analysed using both floatation and Baermann techniques. In order to confirm the microscopic identification of Metastrongylidae first stage larvae, molecular characterization based on the sequence of the ITS-2 was performed. The possible influence of some variables such as the species, sex and age of the animals and the co-infection with other gastrointestinal parasites on the prevalence of cardiorespiratory nematodes was analysed. The most prevalent metastrongylid was Aelurostrongylus abstrusus (15.4%) followed by Angiostrongylus vasorum (4.1%) and Crenosoma vulpis (1.1%). Regarding trichuroids, Eucoleus aerophilus and/or Eucoleus boehmi eggs were detected in 28 dogs (7.7%) and four cats (6.2%). Almost all animals positive to cardiorespiratory nematodes (86.8%) were co-infected with gastrointestinal parasites. The prevalence of Metastrongylidae and respiratory trichuroids was significantly higher in dogs co-infected with Taenia spp. and Toxocara canis or Giardia duodenalis and Sarcocystis spp., respectively. In cats, a significant higher prevalence of Metastrongylidae nematodes was found in animals co-infected with Toxocara cati. Our results reveal that cardiorespiratory nematodes are common in companion animals from north-western Spain, showing higher prevalences than those previously reported from this country. This investigation represents the first report of C. vulpis, E. aerophilus and E. boehmi in dogs from Spain. The identification of a number of zoonotic parasites is of public health concern. Our results indicate that these nematodes should be included in the differential diagnosis of dogs and cats from north-western Spain showing respiratory or cardiac clinical signsThis research was funded by the Program for consolidating and structuring competitive research groups (ED431C 2019/04, Xunta de Galicia, Spain)S

The Age-Related Cryptosporidium Species Distribution in Asymptomatic Cattle from North-Western Spain

An age-related distribution of Cryptosporidium species has been reported in cattle, with C. parvum being predominant in suckling calves, C. bovis and C. ryanae being predominant in post-weaned calves and C. andersoni being predominant in adults. However, variants to this pattern have recently been reported. Thus, fecal samples (n = 594) from asymptomatic cattle were collected in north-western Spain. Animals were classified as 2 years (G5). Cryptosporidium detection and species identification were performed by SSU rRNA PCR. Individual Cryptosporidium prevalence was 16.7%; it significantly decreased with age. Cryptosporidium parvum was predominant in G1 and C. bovis was predominant in the rest of the age classes; C. bovis and C. ryanae were especially prevalent in G2 and G3. Cryptosporidium occultus was not found in suckling calves. Finally, C. andersoni and C. xiaoi were occasionally detected in G5. The presence of C. parvum in all age classes implies significant animal and public health concerns. The predominance of C. bovis in cattle older than 1 month supports the idea that the age-related pattern of Cryptosporidium species described in cattle is not fully consistent, and thus further studies are still needed to identify those factors determining the species distributionThis research was funded by Ministerio de Ciencia e Innovación (Spain), grant number AGL2016-76034-P and by Xunta de Galicia (Spain), grant number 2017-PG117S