Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.     

Pregnancy Diagnosis in Cats Using a Rapid, Bench-top Kit to Detect Relaxin in Urine

Relaxin is a pregnancy-specific hormone in the queen and is produced by the placenta. Both serum and urinary relaxin levels can be used to diagnose and monitor pregnancy in the cat; however, only serum levels are commonly measured in practice. The present study aimed to assess whether urine could be used for the rapid diagnosis of pregnancy at an early stage in domestic cats using a bench-top kit to detect relaxin. Paired serum and urine samples were collected during the first month of gestation in six cats. The samples were tested by applying neat serum, urine or urine diluted in non-pregnant cat serum to the Witness Relaxin kit. Relaxin concentrations in the paired samples were also measured by radioimmunoassay. All undiluted urine samples from pregnant cats tested negative using the bench-top kit; however, the kit was able to detect relaxin in urine after dilution with non-pregnant cat serum. Using this as the test sample, the kit was accurate at diagnosing pregnancy from 28 days after mating and some samples tested positive at 21 days after mating. This preliminary work could lead to the development of a home pregnancy test for cats.     

Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes.

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

Two Unusual Cases of Canine Prostatitis: Prostatitis in a Castrated Dog and Preputial Oedema in an Intact Male

In this study, two unusual presentations of canine prostatitis are described; in the first case a 10‐years‐old neutered Boxer dog was presented to the Veterinary Teaching Hospital of the University of Extremadura with a complaint of anorexia, apathy and preputial discharge. In the second case, a local veterinarian referred an 8‐years‐old male Labrador to the Veterinary Teaching Hospital of the University of Extremadura. The dog had a history of pain in the caudal abdomen and preputial oedema. The final diagnosis in both cases was acute prostatitis. It is concluded that although canine prostatitis is a common disease, sometimes can have presentations that may differ from those classically described in the literature.     

Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation

Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p < 0.05) of this fatty acid in his sperm plasma membranes.