Prevalence, Serotypes, Antimicrobial Resistance and Biofilm-Forming Ability of Listeria monocytogenes Isolated from Bulk-Tank Bovine Milk in Northern Greece.

The prevalence of Listeria monocytogenes in bovine bulk-tank milk (BTM) in Greece has not been previously investigated. The aim of the study was to estimate the prevalence of L. monocytogenes in bovine BTM in Greece and to characterize the isolates in terms of carriage of genes encoding for pathogenic determinants, assess the isolates' biofilm-forming ability and determine their susceptibility against 12 antimicrobials. Samples (n = 138) of bovine BTM were obtained from farms located throughout Northern Greece and were analyzed qualitatively and quantitatively for L. monocytogenes. Five samples (3.6%) tested positive for L. monocytogenes. The pathogen's populations in these positive samples were below 5 CFU/mL. Most isolates belonged to the molecular serogroup "1/2a, 3a". All isolates carried the virulence genes inlA, inlC, inlJ, iap, plcA and hlyA, but actA was detected in only three isolates. The isolates displayed weak to moderate biofilm-forming ability and distinct antimicrobial resistance profiles. All isolates were characterized as multidrug resistant, with resistance to penicillin and clindamycin being a common feature. Considering that L. monocytogenes constitutes a serious public health threat, the key findings of the study, related to the carriage of virulence genes and multidrug resistance, highlight the importance of continued monitoring of the pathogen in farm animals.

Effect of Sugar Beet Pulp and Anionic Salts on Metabolic Status and Mineral Homeostasis during the Peri-Parturient Period of Dairy Sheep.

Sugar beet pulp is a popular by-product of sugar extraction; however, it can potentially cause depletion of Ca availability due to its oxalic content. The experiment examined the effect of sugar beet pulp and anionic salts administration during the dry period on the serum concentration of calcium, magnesium, phosphate, and potassium of dairy sheep. Eighty-seven sheep were divided into three groups (A, B, and C) according to their body condition score (BCS) and age at 40 days before the expected lambing. All groups received alfalfa hay, mixed grass straw, and a concentrate supplement. The concentrate fed to groups B and C contained sugar beet pulp. The nutritional value fed to all three groups was similar, except for Dietary Cation Anion Difference (DCAD). Animals of group A had a DCAD of +198 mEq/kg, animals of group B of +188 mEq/kg, and animals of group C were fed 20 gr/d ammonium chloride to achieve a negative DCAD (-52 mEq/kg). All groups were fed the same ration after lambing. Blood samples were collected 30 d, 20 d, 17 d, 14 d, 10 d, 7 d, and 4 d before lambing (a.p.), 6 h, 12 h, 24 h, 7 d, 10 d, and 15 d after lambing (p.p) for calcium, magnesium, phosphate, and potassium, and 30 d a.p., 7 d, and 15 d p.p. for beta hydroxybutyrate acid (BHBA) concentrations. Urine samples were also collected 20 d, 10 d, 4 d a.p., and 7 d p.p for the evaluation of pH levels. Ca levels of the control group decreased earlier and were lower at 4 d a.p. compared to those of group B and C. Additionally, the control group showed lower p values compared to group C at 20 d and 17 d a.p. P levels recovered earlier post parturition in young (age 1-1.5 years old) compared to older ewes. Group C had lower urine pH values throughout the pre-parturient period, reflecting the acidifying effect of the administered ammonium chloride, without any side effect on macromineral blood concentration. Feeding sugar beet pulp and systemic acidifying before parturition is considered safe and might even be beneficial in preventing hypocalcemia.

Abortion Related to Toxoplasma gondii Infection in a Bactrian Camel (Camelus bactrianus) in Greece: A Case Report.

A female, 5 yr old Bactrian camel was presented to the Exotic and Wildlife Medicine Unit, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, with severe dehydration, depression, anorexia, mild dyspnea and diarrhea. Supportive treatment immediately initiated with fluids, electrolytes and broad-spectrum antibiotics. The general condition of the animal was stable for the next 3 days, but at 4th day became worse, since the camel remained in sternal recumbency, denied to drink water and abortion of a mummified fetus was noticed. The aborted fetus and fetal membranes were submitted for laboratory examinations (bacterial cultures, MZN, cytology, PCR) that revealed Toxoplasma gondii infection. Treatment with sulfadimidine improved the situation of the animal that returned to its farm 1 week later. This seems to be the first reported case in the literature of confirmed toxoplasmic abortion in camels.

Risk factors for Cryptosporidium infection in small ruminants in northern Greece.

The knowledge of risk factors for Cryptosporidium spp. infection in small ruminants is based on limited data. Therefore, the current research aimed to describe the prevalence and risk factors associated with the occurrence of Cryptosporidium infection in sheep and goat herds in northern Greece. Hence, 530 fresh fecal samples from 59 sheep and goat farms were collected and examined for Cryptosporidium oocysts using microscopy of fecal smears stained by the modified Ziehl-Neelsen technique. The overall prevalence of Cryptosporidium infection for both host species was 34% (180/530; 95% confidence interval (CI): 29.9-38). Specifically, the prevalence for sheep and goats was 33.5% (112/334; 95% CI: 28.4-35.6) and 34.7% (68/196; 95% CI: 28-41.4), respectively. Additionally, standardized questionnaires were filled-in to collect data regarding animals' health status, feeding, and other management practices in each farm. In total 22 risk factors hypothesized to be associated with Cryptosporidium infection were investigated. Multiple logistic regression analysis showed that farms with stagnant water were 11.78 (95% CI: 66-61.5) times more likely to be infected with Cryptosporidium than farms without stagnant water (p < 0.05). Furthermore, farms with more than 25% of their animals suffering from diarrhea were 17.39 (95% CI: 3.43-88.3) times more likely to be infected with Cryptosporidium than farms with ≤ 25% of the animals having diarrhea (p < 0.05). These results suggest that the animal health status and the prevailing environmental conditions play an important role in transmitting Cryptosporidium spp. infection.

Brucella spp. distribution, hosting ruminants from Greece, applying various molecular identification techniques.

BACKGROUND: Brucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers. RESULTS: Only one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples. CONCLUSIONS: The present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.

Molecular detection of Brucella spp. in ruminant herds in Greece.

Brucellosis is a worldwide distributed infectious disease. Ruminants and other animal species (swine, dogs, equids, etc.), as well as wild mammals, can be affected. The disease can be transmitted to humans through the food chain or by direct contact with infected animals. Because of the relatively high economic burden due to abortions within a herd, significant efforts have been employed and hence the disease in most European countries has been eradicated. Accordingly, Greece applies both control and eradication programs concerning small ruminants (sheep and goats) and bovines depending on the geographical area. Current challenges in the standard antibody-based laboratory methods used for Brucella detection are the failure to differentiate antibodies against the wild strain from the ones against the vaccine strain Rev1 and antibodies against B. melitensis from those against B. abortus. The aim of the study was to reexamine and combine previously published protocols based on PCR analysis and to generate a rapid, not expensive, and easy to perform diagnostic tool able to confirm the doubtful results delivered from serology. For this reason, 264 samples derived from 191 ruminants of the farm and divided in 2 groups (male/female) were examined with a modified DNA extraction and PCR protocol. Molecular examination revealed the presence of Brucella spp. in 39 out of 264 samples (derived from 30 animals). In addition, Brucella spp. was detected in infected tissues such as testicles, inguinal lymph nodes, fetal liver, and fetal stomach content.

Mycoplasma agalactiae ST35: a new sequence type with a minimal accessory genome primarily affecting goats.

BACKGROUND: Mycoplasma agalactiae, causing agent of contagious agalactia, infects domestic small ruminants such as sheep and goats but also wild Caprinae. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes spreading rapidly in an infected herd. RESULTS: In an outbreak of contagious agalactia in a mixed herd of sheep and goats, 80% of the goats were affected displaying swollen udders and loss of milk production but no other symptom such as kerato-conjunctivitis, arthritis or pulmonary distress commonly associated to contagious agalactia. Surprisingly, none of the sheep grazing on a common pasture and belonging to the same farm as the goats were affected. Whole genome sequencing and analysis of M. agalactiae strain GrTh01 isolated from the outbreak, revealed a previously unknown sequence type, ST35, and a particularly small, genome size of 841'635 bp when compared to others available in public databases. Overall, GrTh01 displayed a reduced accessory genome, with repertoires of gene families encoding variable surface proteins involved in host-adhesion and variable antigenicity being scaled down. GrTh01 was also deprived of Integrative Conjugative Element or prophage, and had a single IS element, suggesting that GrTh01 has a limited capacity to adapt and evolve. CONCLUSIONS: The lack of most of the variable antigens and the Integrative Conjugative Element, both major virulence- and host specificity factors of a M. agalactiae strain isolated from an outbreak affecting particularly goats, indicates the implication of these factors in host specificity. Whole genome sequencing and full assembly of bacterial pathogens provides a most valuable tool for epidemiological and virulence studies of M. agalactiae without experimental infections.

Polymorphisms of Codons 110, 146, 211 and 222 at the Goat PRNP Locus and Their Association with Scrapie in Greece.

Scrapie is considered an endemic disease in both sheep and goats in Greece. However, contrary to sheep, in goats more than one prion protein (PrP) polymorphism has been recognized as a candidate for resistance breeding against the disease. For an impression, candidates which are circulating, (i) brain samples (n = 525) from scrapie-affected (n = 282) and non-affected (n = 243) animals within the national surveillance program, and (ii) individual blood samples (n = 1708) from affected (n = 241) and non-affected (n = 1467) herds, in a large part of mainland Greece and its islands, were collected and assayed. A dedicated Taqman method was used to test for amino acid polymorphisms 110T/P, 146N/S/D, 211R/Q, and 222Q/K. Highly prevalent genotypes were 110TT, 146NN, 211RR, and 222QQ. The frequencies of polymorphisms in blood and negative brain samples for codons 110P, 211Q, and 222K were 4.0%, 3.0%, and 1.9%, respectively, while 146D (0.7%) was present only on Karpathos island. Codon 110P was exclusively found in scrapie-negative brains, and homozygous 110P/P in two scrapie-negative goats. It is concluded that breeding programs in Karpathos could focus on codon 146D, while in other regions carriers of the 110P and 222K allele should be sought. Case-control and challenge studies are now necessary to elucidate the most efficient breeding strategies.

Coxiella burnetii Shedding in Milk and Molecular Typing of Strains Infecting Dairy Cows in Greece.

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece.

A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLVs) are highly diverse retroviruses infecting sheep and goats. Although PCR-based testing is being utilized for diagnostics, its application is hampered by various factors. These include, among others, the exceptionally high genetic variability of SRLVs, as well as the low number of infected blood monocytes. For this reason, a highly sensitive and specific semi-nested real-time PCR for proviral DNA detection and quantification was developed. The method is innovative in that a) its design is based on selecting the preferred codon usage in the targeted conserved genomic regions and b) oligospermine-conjugated degenerate primers with increased Tm were utilized. Modifications permitted primer/template duplex formation in the cases of mismatches due to sporadic nucleotide polymorphisms in a number of variant SRLV strains and consequently, the detection of highly diverse SRLV strains. The potential loss of analytical sensitivity and specificity was counterbalanced by including a semi-nested step in combination with LNA probes. An in silico procedure for the evaluation of hybridization efficiency of the designed oligonucleotides to all known targeted variants was also implemented. The method presents a linear range of quantification over a 3-log10 range and a limit of detection of 3.9 proviral dsDNA copies per reaction. Its diagnostic performance was evaluated by testing field samples from seropositive and seronegative animals, followed by phylogenetic analysis of the strains detected. To further increase the diagnostic sensitivity, a DNA extraction protocol for blood leukocytes was developed and evaluated. A minimum of 500 ng input DNA is recommended for PCR-based detection of SRLV proviral DNA, given the low numbers of infected blood monocytes. The developed methodology may serve as a useful tool, which can be adjusted for the quantitative detection of viruses exhibiting high genetic variability.

Effect of experimental nematode infection on serum mineral concentrations in growing lambs.

The objective of the present study was to determine the possible effect of gastrointestinal nematodes upon serum mineral concentrations of lambs. Twelve male lambs were used. Lambs were randomly assigned to 2 groups (n = 6): Group 1 infected with gastrointestinal nematodes and Group 2 as controls. Lambs of Group 1 were infected with a single dose of 15,000 L3 larvae of GI nematodes (Haemonchus, Teladorsagia, Trichostrongylus, Cooperia and Oesophagostomum-Bunostomum). Blood samples were collected from the investigated animals individually every 2 weeks. However, the differences in serum macro-minerals (Ca, Mg, P, K, and Na) among groups were not significant. Although the differences in serum macro-minerals among groups were not significant and the iron serum concentration remained unaltered, the gastrointestinal parasitism reduced significantly/substantially the serum copper levels.

Development of real-time PCR-based methods for the detection of enzootic nasal tumor virus 2 in goats.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.

Genotyping of Coxiella burnetii in sheep and goat abortion samples.

BACKGROUND: Q fever, caused by Coxiella burnetii, is a zoonosis that presents a worldwide distribution and affects both humans and animals. The route of dispersal of the pathogen by ruminants into the environment usually involves stages of abortion and parturition, nevertheless the agent can, also, be detected in other animal samples. Therefore it is considered as important in terms of proper diagnosis, as well as, for epidemiology and surveillance purposes, to genotype the pathogen. The aim of the current study was to investigate the presence of different genotypes of the agent in animals that had suffered from abortion during a two-year survey in Greece. RESULTS: Sixty nine tissue samples (37 stomach contents, 11 liver samples, 21 cotyledons) were collected from 59 abortion cases in sheep (N = 45) and goats (N = 14) from 65 farms at eight different areas of Greece. Samples were screened by qPCR and positive ones were further genotyped using a 10-locus multiple loci (ms 1, 3, 7, 12, 20, 21, 22, 26, 30 and 36) variable number of tandem repeat analysis (MLVA) method. Three genotypes were identified in sheep (A, B, C). Samples representing each of the obtained MLVA profile were further used for MST genotyping. Ten spacers (Cox 2, 5, 6, 18, 20, 22, 37, 51, 56 and 57) were amplified. A close relatedness among the identified MLVA genotypes was confirmed since they all belonged to MST group 32. CONCLUSIONS: The current study introduces into the aspect of genotyping of C. burnetii in Greece. Further studies are needed to explore the presence of more genotypes, to associate the genotypes circulating in the animal and tick population with those causing human disease in order to further expand on the epidemiological aspects of the pathogen.

Genotypes and subtypes of Cryptosporidium spp. in diarrheic lambs and goat kids in northern Greece.

Inconsistent data exist on the distribution of zoonotic Cryptosporidium species and subtypes in sheep and goats in European countries, and few such data are available from Greece. In this study, 280 fecal specimens were collected from 132 diarrheic lambs and 148 diarrheic goat kids aged 4 to 15 days on 15 farms in northern Greece, and examined for Cryptosporidium spp. using microscopy of Ziehl-Neelsen-stained fecal smears. Cryptosporidium spp. in 80 microscopy-positive fecal specimens (39 from lambs and 41 from goat kids) were genotyped by PCR-RFLP analysis of the small subunit rRNA gene and subtyped by sequence analysis the 60 kDa glycoprotein gene. Among the 33 specimens successfully genotyped, C. parvum was found in 32 and C. xiaoi in one. Seven subtypes belonging to two subtype families (IIa and IId) were identified among the 29 C. parvum specimens successfully subtyped, including IIaA14G2R1 (1/29), IIaA15G2R1 (6/29), IIaA20G1R1 (7/29), IIdA14G2 (1/29), IIdA15G1 (9/29), IIdA16G1 (3/29), and IIdA23G1 (2/29). Lambs were more commonly infected with C. parvum IIa subtypes, whereas goat kids were more with IId subtypes. The results illustrate that C. parvum is prevalent in diarrheic lambs and goat kids in northern Greece and these animals could potentially play a role in epidemiology of human cryptosporidiosis.

Isolation and whole-genome sequencing of a Crimean-Congo hemorrhagic fever virus strain, Greece.

Crimean-Congo hemorrhagic fever virus (CCHFV) was isolated from a pool of two adult Rhipicephalus bursa ticks removed from a goat in 2015 in Greece. The strain clusters into lineage Europe 2 representing the second available whole-genome sequenced isolate of this lineage. CCHFV IgG antibodies were detected in 8 of 19 goats of the farm. Currently CCHFV is not associated with disease in mammals other than humans. Studies in animal models are needed to investigate the pathogenicity level of lineage Europe 2 and compare it with that of other lineages.

Epidemiological characteristics and clinicopathological features of bluetongue in sheep and cattle, during the 2014 BTV serotype 4 incursion in Greece.

During 2014, an outbreak of Bluetongue virus (BTV) infections attributed to serotype 4 occurred in Greece and spread to south-eastern Europe. In the present article, the clinical and epidemiological data of 15 sheep flocks and 5 dairy cattle herds affected in Greece are described. In sheep, the most frequent clinical signs observed were fever, hyporexia, and edema of the face. A number of clinically affected sheep had chronic laminitis resulting in chronic lameness. Confirmation of suspect clinical cases was performed using BTV-specific real-time RT-PCR, and serotype 4-specific RT-PCR. The average morbidity of bluetongue in the sheep flocks was estimated to be 15.3 % (95 % C.I. 6.8-23.8 %) and the average mortality and case fatality were 4.5 % (95 % C.I. 1.5-7.6 %) and 32.0 % (95 % C.I. 18.1-42.9 %), respectively. The BTV seroprevalence and the ratio of clinical manifestations-to-infections determined in seven of these flocks, were on average 36.5 % (95 % C.I. 15.7-57.3 %) and 24.6 % (95 % C.I. 12.8-36.3 %). BTV ratio of clinical manifestations-to-infections was higher in the imported western European sheep breeds examined compared to the local ones. In dairy cattle, the average herd prevalence of viremia was 48.8 % (95 % C.I. 15.3-82.4 %) and none had signs associated with bluetongue. The results of this study indicate that the 2014 Greek BTV-4 has significant impact on the health status and the viability of sheep in affected flocks but does not cause clinical signs in cattle, despite the high prevalence of viremia.

Genetic characterisation of Taenia multiceps cysts from ruminants in Greece.

This study was designed to genetically characterise the larval stage (coenurus) of Taenia multiceps from ruminants in Greece, utilising DNA regions within the cytochrome c oxidase subunit 1 (partial cox1) and NADH dehydrogenase 1 (pnad1) mitochondrial (mt) genes, respectively. A molecular-phylogenetic approach was used to analyse the pcox1 and pnad1 amplicons derived from genomic DNA samples from individual cysts (n=105) from cattle (n=3), goats (n=5) and sheep (n=97). Results revealed five and six distinct electrophoretic profiles for pcox1 and pnad1, respectively, using single-strand conformation polymorphism. Direct sequencing of selected amplicons representing each of these profiles defined five haplotypes each for pcox1 and pnad1, among all 105 isolates. Phylogenetic analysis of individual sequence data for each locus, including a range of well-defined reference sequences, inferred that all isolates of T. multiceps cysts from ruminants in Greece clustered with previously published sequences from different continents. The present study provides a foundation for future large-scale studies on the epidemiology of T. multiceps in ruminants as well as dogs in Greece.

Regurgitations in a Lamb with Acute Coenurosis-A case Report.

Coenurosis is a disease of the central nervous system in sheep, caused by Coenurus cerebralis, the larval stage of Multiceps multiceps, which inhabits the small intestine of Canidae. A case of regurgitations in a 2.5 month old lamb with acute coenurosis is being reported. The lamb was presented with a sudden onset of ataxia and regurgitations for 10 days. The post-mortem examination revealed 4 immature C. cerebralis cysts between 0.5 and 1.5 cm in diameter located in the brainstem and cerebellum, and histopathological examination revealed multifocal pyogranulomatous meningoencephalitis, so a diagnosis of acute coenurosis was established. Thus, acute coenurosis should be included in the differential diagnosis of regurgitations in lambs.

Epidemiological Observations on Cryptosporidiosis in Diarrheic Goat Kids in Greece.

This study aimed at investigating the occurrence of Cryptosporidium spp. in diarrheic goat kids in Greece and the risk factors associated with cryptosporidiosis. Altogether, 292 diarrheic 4-15-day-old goat kids from 54 dairy goat herds of Northern Greece were examined. Oocysts of Cryptosporidium spp. were detected in 223 of 292 (76.4%) goat kids and the intensity of infection was scored as "high" in 142 samples, "moderate" in 45 samples, and "low" in 36 samples. Larger herds (>200 animals) had higher infection rates than smaller ones, although this difference was not statistically significant. Significantly higher infection rates were observed in herds during late kidding season (1 January to 30 April) compared to the early one (1 September to 31 December). These results suggest that cryptosporidiosis is very common in diarrheic goat kids in Greece, especially in large herds during the late parturition season.

In vitro susceptibilities of caprine Mycoplasma agalactiae field isolates to six antimicrobial agents using the E test methodology.

The minimum inhibitory concentrations (MICs) of enrofloxacin, ciprofloxacin, spectinomycin, tetracycline, spiramycin and erythromycin against 30 caprine Greek isolates of Mycoplasma agalactiae were determined using E test methodology. The E test strips were placed on Eaton's agar medium without antimicrobials and phenol red. MICs were then read by determining where the growth inhibition zone intersected with the MIC scale on the strip. An MIC value of 8 µg/mL was considered as a guide to mycoplasma resistance. All isolates were sensitive to fluoroquinolones (MIC50, 0.19 g/mL; MIC90, 0.38 µg/mL; highest MIC, 0.5 µg/mL), spectinomycin (MIC50, 0.5 µg/mL; MIC90, 1 µg/mL; highest MIC, 1 µg/mL), and spiramycin (MIC50, 1 µg/mL; MIC90, 1.5 µg/mL; highest MIC, 2 µg/mL). Two strains exhibited resistance to tetracycline (MIC 32 µg/mL) but these were not found to carry any of the tet(M), tet(O), and tet(S) resistance genes. Finally all isolates expressed resistance to erythromycin (MIC50, 128 µg/mL; MIC90, >256 µg/mL).