Extensive Countrywide Molecular Identification and High Genetic Diversity of Haemonchus spp. in Domestic Ruminants in Greece.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

Helminth Infections in Dairy Sheep Found in an Extensive Countrywide Study in Greece and Potential Predictors for Their Presence in Faecal Samples.

The aims of the present study were: (a) to describe the prevalence of helminth infections from pooled faecal samples from sheep flocks across Greece and (b) to evaluate flock-related factors potentially associated with the presence of these infections in the flocks. An extensive countrywide study was performed on 325 sheep farms throughout Greece; faecal samples were collected from ewes and processed for the identification of helminth parasites. Helminths were detected in samples from 92.9% of flocks; these included Dicrocoelium dendriticum (16.7% of flocks), Fasciola hepatica (0.6%), Paramphistomum cervi (2.2%), Moniezia spp. (18.8%), Trichostrongylidae (85.5%), Nematodirus spp. (18.8%), Strongyloides papillosus (7.1%), Trichuris spp. (20.0%) and lungworms (17.8%). Mean Trichostrongylidae counts across all flocks in the study were 215 epg. Specifically, for Trichostrongylidae, there were differences between flocks that had or had not received anthelmintics during the two months prior to sampling, as well as between flocks located in various areas of the country. In multivariable analyses, for the outcome 'high (>300) epg counts in faecal samples', the month into the lactation period at sampling and the application of reproductive control practices on the farm emerged as significant factors; for the outcome 'high proportion (>63%) of Teladorsagia spp. in faecal samples', the availability of straw bedding emerged as a significant factor; and for the outcome 'high proportion (>63%) of Haemonchus contortus in faecal samples', the age of the farmer emerged as a significant factor. For the outcome 'presence of Trichuris spp. in faecal samples', the provision of finished feed (concentrate) to animals emerged as a significant factor, whilst, finally, for the outcomes 'presence of D. dendriticum in faecal samples' and 'presence of lungworms in faecal samples,' no significant factors emerged.

Deltamethrin Application on Pre-Weaned Calves Improves Feed Consumption, Stress and Fatigue Status under Heat Stress Conditions.

Fly infestation remains a universal problem for dairy cattle herds, affecting the animals' health and welfare status. Pre-weaned dairy calves are significantly challenged by the direct and indirect consequences of severe fly infestation, heat-stress and their interaction, which contribute to a stressful and fatiguing environment. Among several physiological, behavioral, clinical and biochemical traits, serum cortisol (SC) and creatine kinase (CK) levels, as well as feed consumption can be used as valid indicators of potential stressful and fatiguing conditions and, therefore, can be efficiently used for stress analysis studies. Hence, the objective of the study was to assess the fly-repellency effect of deltamethrin on pre-weaned dairy calves exposed to heat stress conditions, as well as its association with SC, CK concentrations and feed consumption. Two commercial dairy cattle herds of the Holstein breed in Central Macedonia (Greece) were involved in the study during summer months and under heat stress conditions. Deltamethrin administration resulted in (i) a decreased fly population (100% Musca domestica) landing on pre-weaned dairy calves, (ii) a reduced SC (stress indicator) and CK (fatigue indicator) concentration, and (iii) an increased consumption of feedstuff in deltamethrin treated animals compared to the untreated ones.

Protozoan Parasites in Adult Dairy Small Ruminants and Potential Predictors for Their Presence in Faecal Samples.

There is a scope to study protozoan infections in adult ewes and does, as these animals can act as reservoirs of infection for lambs and kids, for which these pathogens are harmful. The objectives of this study were to describe the prevalence of protozoan infections in faecal samples from adult sheep and goats on dairy farms across Greece and to evaluate farm-related factors potentially associated with the presence of protozoan infections in these animals. A cross-sectional study was performed on 325 sheep and 119 goat farms throughout Greece; faecal samples were collected from ewes and does and processed for the identification of protozoan parasites. Eimeria oocysts were found in faecal samples from 69% of farms (72% of sheep farms and 61% of goat farms), Giardia cysts in samples from 33% of farms (33% of sheep farms and 34% of goat farms) and Cryptosporidium oocysts in samples from 8% of farms (7% of sheep farms and 11% of goat farms). In a multivariable analysis, for the presence of Eimeria in samples from sheep farms, the lack of a designated building for lambs emerged as a significant factor; for the presence of Giardia in samples from goat farms, the availability of a main building for animals emerged as a significant factor; for the presence of Cryptosporidium, the lack of grazing and the management system emerged as the main significant factors in sheep and goat farms, respectively. Protozoa were found significantly more frequently in samples collected from farms on which farmers considered diarrhoea as an important health problem in their lambs/kids.

Haemonchus contortus Parasitism in Intensively Managed Cross-Limousin Beef Calves: Effects on Feed Conversion and Carcass Characteristics and Potential Associations with Climatic Conditions.

The objectives of the study were: (a) to study the effect of Haemonchus spp. on the growth performance characteristics of fattening calves, (b) to assess any potential effects on carcass characteristics and (c) to investigate the potential role of climatic conditions in the process of the infection. The study was conducted for 201 days in an intensively managed cross-Limousin herd. The animals were divided into two equal groups: those receiving anthelmintic treatment (AT) and the untreated (C) controls. The same nutritional regime was applied to both groups and the feed consumption was calculated daily. Standard parasitological examinations were performed at weekly intervals. At slaughter, carcasses were weighed and assessed for conformation and fat cover classes. Climatic variables were obtained for the location of the farm and a temperature-humidity index was calculated. Before anthelmintic treatment with ivermectin, there was no difference in parasitic burdens between the two groups: 544 (AT) vs. 554 (C) epg, whilst after it, counts were 0 and 450-700 epg, respectively, with over 96% of larvae identified as Haemonchus spp. and, later, confirmed as Haemonchus contortus. It was concluded that treated animals had a higher average total bodyweight gain, higher feed conversion ratio and carcass yield of superior quality than controls. There was a difference between the two groups in the pattern of reduction of dry matter intake as the temperature-humidity index increased.

Serological Assessment of Ascaris suum Exposure in Greek Pig Farms and Associated Risk Factors Including Lawsonia intracellularis.

The effects of nematodes and bacteria on intestinal health are of primary importance in modern swine production. The aim of the present study was to assess the seropositivity status of Ascaris suum infection in fatteners in intensive swine farms in Greece and address possible risk factors, including Lawsonia intracellularis as a predisposing factor to swine ascariosis. In total, 360 blood serum samples from pigs in the late fattening period, from 24 Greek swine farrow-to-finish farms (15 samples/farm) were collected and tested with Svanovir® A. suum antibody ELISA and Ileitis antibody ELISA. The results demonstrated 34.4% seropositive samples for A. suum and 42.2% for L. intracellularis. The analysis of predisposing risk factors suggested that the frequency of application of anthelminthic treatment to sows more than two times per year was significantly associated with the lower likelihood of A. suum infection, whereas a greater likelihood of A. suum infection was observed in pigs with concurrent L. intracellularis exposure. The results highlight the importance of proper anthelminthic metaphylaxis of the breeding stock, as well as the likely outcome of concurrent exposure to two intestinal pathogens in pigs, implying a possible association between intestinal nematodes and bacteria in swine.

High Milk Somatic Cell Counts and Increased Teladorsagia Burdens Overshadow Non-Infection-Related Factors as Predictors of Fat and Protein Content of Bulk-Tank Raw Milk in Sheep and Goat Farms.

This paper presents the results of an extensive countrywide investigation performed in 325 dairy sheep flocks and 119 goat herds throughout Greece. The objectives of the study were (a) to investigate fat and protein content in the bulk-tank raw milk of small ruminant farms in Greece and (b) to identify factors potentially influencing that content and factors that can contribute to increased content. The mean fat/protein contents in bulk-tank raw milk of sheep and goats were 6.16 ± 0.05%/4.43 ± 0.01% and 4.77 ± 0.44%/3.23 ± 0.30%, respectively. Significant differences were seen in protein content between farms in the various parts of the country. For sheep, multivariable analyses revealed breed and age of lamb removal from dams as significant for fat content, and somatic cell counts, management system in the farm, administration of anthelmintic treatment during the last stage of pregnancy, and farmer education as significant for protein content. For goats, significant factors were month into lactation period, age of kid removal from dams, and breed for fat content, and somatic cell counts, month into lactation, grazing duration, and % Teladorsagia larvae in faecal samples for protein content. For concurrently high fat and protein content, in multivariable analyses, the following emerged as significant factors: somatic cell counts in milk, numbers of parasite eggs in faeces, and veterinary collaboration (sheep), and month into lactation and somatic cell counts in milk (goats). The results indicate that high somatic cell counts in milk (reflecting the presence of mastitis) and gastrointestinal parasitic infections (mainly Teladorsagia infection) appear to exert a more significant influence on fat and protein content of milk, in comparison to non-infection-related factors.

Haemonchosis: A Challenging Parasitic Infection of Sheep and Goats.

The paper reviews the challenges about haemonchosis-a significant and common parasitic infection of small ruminants. Haemonchus contortus is a highly pathogenic parasite that localises in the abomasum of affected animals and exerts its pathogenicity by blood-sucking activity, adversely affecting the health and productivity of animals. The first challenge is the uneven distribution of the infection globally, this being more prevalent in tropical and subtropical and warm temperate and summer rainfall regions than in cool and cold temperate and arid regions; hence, this leads in differences in the approaches required for its control. Another challenge is the widespread presence of Haemonchus strains resistant to the various anthelmintics available: Benzimidazoles, imidazothiazoles, macrocyclic lactones, closantel and monepantel, which makes the control of the infection difficult. The third challenge refers to the difficulty of diagnosing the disease, given that field evidence can provide suspicion about the infection, which needs to be subsequently confirmed by laboratory tests through parasitological or molecular techniques. The final challenge relates to the difficulties in the control of the infection and the necessity to use pharmaceutical products cautiously and with a planned approach, to avoid further development of anthelmintic resistance, also given that use of a recently licenced vaccine is not widespread. In conclusion, at the moment, we should be concerned, but not worried, about this infection, and apply correctly the appropriate health management plans.

Will Fly Repellency Using Deltamethrin Reduce Intramammary Infections, Stress and Fatigue Indicators of Dairy Ewes under Intensive Management?

Intramammary infections (IMIs) caused by various pathogens may lead to clinical or subclinical mastitis, challenging the health and welfare status of infected animals and decreasing the quantity and quality of the produced milk. Additionally, the zoonotic potential of some of the pathogens isolated from IMI cases, the emergence of antibiotic resistance due to the extensive antibiotic use for IMI treatment, and the accumulation of antibiotic residues in milk and meat represent significant concerns for public health. Therefore, the investigation of IMI risk factors and the proposal of efficient measures to mitigate their effects on animal health and welfare is crucial. Although fly infestation is considered to play a significant role in the transmission of IMI pathogens, its adverse effects on udder health and the overall comfort status of dairy ewes have not been quantified and assessed on an evidential basis. Hence, the objectives of this study were to assess, for the first time, the fly repellent effect of deltamethrin and link it to: (i) the occurrence of common bacterial IMI; (ii) the somatic cell counts in milk; and (iii) the serum cortisol and creatine kinase levels (stress and fatigue indicators). The study was carried out in an intensive dairy sheep farm in northern Greece, during peak fly season. Deltamethrin treatment was associated with a reduced (i) number of flies (mostly Musca domestica) landing on treated ewes, compared to untreated ones (p < 0.05); (ii) colony-forming units in the case of Non-aureus Staphylococci IMIs (p < 0.05); and (iii) number of somatic cells in the milk (p < 0.001). Finally, serum cortisol and creatine kinase levels were significantly lower in deltamethrin-treated ewes (p < 0.001), indicating a less stressful environment for them.

Rapid on-site diagnosis of canine giardiosis: time versus performance.

BACKGROUND: Infections by protozoans of the genus Giardia are a common cause of diarrhea in dogs. Canine giardiosis constitutes a disease with a zoonotic potential; however, it is often underestimated due to its challenging diagnosis. The objective of the study was to assess the diagnostic performance of an immunochromatographic strip test (SpeedTM Giardia, Virbac, France) comparing it with microscopy (zinc sulfate flotation) by utilizing the combination of an enzyme immunoassay (ProSpecTTM Giardia EZ Microplate Assay, Oxoid Ltd., UK) and the PCR as the gold standard. A positive result in both ELISA and PCR was set as the gold standard. METHODS: Initially, fecal samples from dogs with clinical signs compatible with giardiosis were tested with the SpeedTM Giardia test and separated into two groups of 50 samples each: group A (positive) and group B (negative). Thereafter, all samples were examined by zinc sulfate centrifugal flotation technique and assayed by the ProSpecTTM Giardia Microplate Assay and PCR. The performance of the SpeedTM Giardia and zinc sulfate centrifugal flotation tests were calculated estimating sensitivity, specificity, and positive and negative likelihood ratio; the chi-square and McNemar tests were used for the comparison of the two methods. RESULTS: Giardia cysts were not detected by microscopy in 16 out of the 50 samples (32%) of group A and in none of group B samples. Eight out of 50 samples in group B (16%) were tested positive both with the ProSpecTTM Giardia Microplate Assay and PCR. Fecal examination with the SpeedTM Giardia test was more sensitive (86.2%) than the parasitological method (58.6%, P < 0.001) while the specificity of both methods was 100%. CONCLUSIONS: The SpeedTM Giardia test is an easy-to-perform diagnostic method for the detection of Giardia spp., which can increase laboratory efficiency by reducing time and cost and decrease underdiagnosis of Giardia spp. infections. This immunochromatographic strip test may be routinely exploited when a rapid and reliable diagnosis is required, other diagnostic techniques are unavailable and microscopy expertise is inefficient. In negative dogs with compatible clinical signs of giardiosis, it is recommended either to repeat the exam or proceed with further ELISA and PCR testing.