Subclinical mastitis in Lacaune sheep: Causative agents, impacts on milk production, milk quality , oxidative profiles and treatment efficacy of ceftiofur.

Mastitis is a major disease affecting dairy sheep. It is caused by microorganisms that generate inflammation of the mammary gland in response to tissue invasion. This syndrome affects the welfare of ewes, as well as the production and quality of the milk, thereby reducing its productive efficiency. Because mastitis causes inflammation process, it also increases the production of free radicals that cause lesions via lipoperoxidation, causing damage to proteins, cells and tissues. One way to minimize the impact of the disease is antimicrobial treatment. Nevertheless, the continuous use of antimicrobials contributes to microbial resistance, in addition to producing residues in the milk and derivatives if not given during the grace period. Therefore, the objective of this study was to evaluate the consequences of subclinical mastitis on ewe health, milk production, milk composition and quality. We also evaluated the susceptibility of the bacteria in vitro using disk diffusion antibiograms. Finally, we performed two-way testing of efficacy of treatment in Lacaune ewes using the same agents. In the first stage of the study, 30 lactating ewes (±90 days) were used, 10 of which were negative on the CMT (California Mastitis Test) used as control group (CG) and 20 sheep with subclinical mastitis diagnosed by CMT (MG). Samples were collected and several analyses were performed on the milk and blood. We found that ewes in the MG had higher lipid peroxidation in serum and milk, as well as lower production, with reduction of the total dry extract in milk. There were 15 isolates of Staphylococcus hyicus, four isolates of each S. epidermidis and S. intermedius, and two isolates of Corynebacterium spp. The primary hematological result was leukocytosis in ewes with mastitis. Based on the antibiogram, we chose ceftiofur for in vivo tests. In this stage, we divided the sheep with subclinical mastitis into two subgroups of 10 ewes each, to receive drug by two routes: intramuscular (IM) and intramammary (IMM). In the IMM group, of the 10 CMT-positive ewes at the beginning of the experiment, seven were already negative by the racket test 120 h after the last application (70% efficacy). In the IM group, of the 10 positive ewes, only four were negative after 120 h of the final application, a low efficacy treatment (40%). We evaluated antimicrobial residues in the milk of treated animals. We found this material within 5 days after treatment in the two forms used; despite the fact that the product's stated withholding period is 3 days. We conclude that ewes with mastitis produce less milk of lower quality. We also conclude that, although ceftiofur is 100% effective in vitro, when used in ewes with mastitis, the efficacy did not exceed 70%, and was more efficient when administered via the intramammary route.

New sequence type ST3756 of Staphylococcus aureus subspecies anaerobius as the causative agent of abscessing lymphadenitis in sheep.

Morel's disease is a form of abscessing lymphadenitis of sheep and goats caused by Staphylococcus aureus subspecies anaerobius. In Europe and Africa, the disease is linked to S. aureus of multilocus sequence type 1464. In an outbreak recorded in 2015 in a flock of 530 animals in the district of Nymburk, Czech Republic, Europe, the causative agent was cultured and subsequently confirmed by Maldi-TOF. Neither antibiotic therapy nor surgical interventions met any success, although the strain isolated was found to be sensitive to antibiotics used. Vaccination and revaccination with inactivated autogenous vaccine administered subcutaneously was relatively successful. Subsequent multilocus sequence typing revealed the presence of new S. aureus sequence type 3756, different from 1464 in three out of seven genes typed. The isolate thus represents a new sequence type of Staphylococcus aureus ssp. anaerobius which should be considered as a causative agent of Morel's disease.

Pasteurella multocida inactivated with ferric chloride and adjuvanted with bacterial DNA is a potent and efficacious vaccine in Balb/c mice.

PURPOSE: Pasteurella multocida (P. multocida) is a principal pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of pneumonia and haemorrhagic septicaemia in cattle, sheep and goats, fowl cholera in chickens and progressive atrophic rhinitis in swine. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with bacterial DNA (IIV+bDNA) as an adjuvant in mice. METHODOLOGY: P. multocida was grown in BHI broth, inactivated with formalin and FeCl3 and adjuvanted with alum and bDNA. Mice were immunized with two whole-cell inactivated vaccine doses 2 weeks apart. The animals were challenged 4 weeks after booster immunization. Immunogens (vaccines and bDNA) posed no safety problems when mice were injected subcutaneously (s/c) with these preparations. The serum antibody titres were tested by ELISA. At 28 days post immunization, cell-mediated immunity responses were determined. The responses were measured by assay of IL-6 and IL-12 in lymphocyte spleen culture supernatants. RESULTS: ELISA results showed that the levels of antibodies in iron inactivated with bDNA adjuvant groups were higher than in the formalin inactivated with alum adjuvant vaccine group. The protection rate of IIV+bDNA adjuvant vaccine was superior to that of the other vaccines and it protected 100 % of the challenge group mice. Following immunization, bDNA promoted increased production of interleukins compared to the control groups. CONCLUSION: These studies indicate that bDNA is effective as an immune adjuvant, and along with stimulatory bDNA represent promising new humoral and cellular immune enhancers for vaccination applications. In addition, this vaccine is able to provide long-term protection against infection.

Understanding the health and production impacts of endemic Chlamydia pecorum infections in lambs.

Lamydia pecorum is a globally recognised livestock pathogen that is capable of causing severe and economically significant diseases such as arthritis in sheep and cattle. Relatively little information is available on the clinical progression of disease and the long-term effects of asymptomatic and symptomatic chlamydiosis in sheep. Recent studies in calves indicate that endemic C. pecorum infections may reduce growth rates. To investigate the clinical health parameters and production impacts of endemic C. pecorum infection in an Australian commercial lamb flock, we performed bimonthly sampling and clinical health assessments on 105 Border Leicester lambs from two to ten months of age. Chlamydial status was investigated via serology and species-specific quantitative PCR. Throughout the study period, conjunctivitis remained a persistent clinical feature while signs of arthritis (e.g. palpable synovial joint effusions) resolved in a subset of lambs while persisting in others. Clinical disease and C. pecorum infection were highest at six months of age (weaning). As previously reported, peak seroconversion tends to occur two months after the onset of clinical symptoms (6 months of age), with lambs clearing chlamydial infection by 10 months of age, despite ongoing disease still being present at this time. Notably, the presence of chlamydial infection did not affect lamb mass or growth rates throughout the study. At necropsy, C. pecorum was not detected within the joints of lambs with chronic arthritis. Molecular analysis of the strains in this flock suggest that the infecting strains circulating in this flock are clonal C. pecorum pathotypes, denoted ST 23, commonly associated with conjunctivitis and polyarthritis in Australian sheep. This study provides a platform for further research in the epidemiology and disease transmission dynamics of C. pecorum infections in sheep.

Report: Prevalence and antibiotic trials against Salmonella enterica isolated from diarrheic lambs and kids.

Salmonella enterica (S. enterica) is the major zoonotic threat for small ruminants and humans responsible for huge economic losses and high mortality in Pakistan. Lambs and kids of Lahore district were examined to determine the prevalence, hematology and chemotherapy of S. enterica. A total of 200 diarrheic samples (n=100 lambs; n=100 kids) were collected and examined; 59 (29.50%) were found positive for S. enterica. Lambs had lightly greater prevalence (31%) than kids (29%). The frequency analysis (OR=1.16 [reciprocal =0.87]) showed non-significant difference in both the lambs and kids. The significant decrease (Pℜ0.001) in hemoglobin, pack cell volume and total erythrocyte count was observed in infected lambs and kids. Results of in-vitro antibiotic susceptibility test revealed that S. enteric isolated from both lambs and kids were susceptible to levofloxacin, ciprofloxacin, ofloxacin, gentamicin, azithromycin, tobramycin, amoxicillin, ampicillin and nalidixic acid. Where as the results of in vivo antibiotic trials showed that isolates from both lambs and the kids with diarrhea were susceptible to levofloxacin and ciprofloxacin.

A study on ovine pneumonic pasteurellosis: isolation and identification of Pasteurellae and their antibiogram susceptibility pattern in Haramaya District, Eastern Hararghe, Ethiopia.

BACKGROUND: Sheep constitute the second major component of livestock in Ethiopia. However, efficient utilization of this potential resource is hampered by combination of health problems, poor management and feed shortage. Haramaya district is one of the remote settings in Ethiopia where information about the livestock disease is not well documented. Hence this study was conducted to determine the causative agents and their antimicrobial susceptibility pattern of bacterial Pasteurella isolates among pneumonic ovine in Haramaya district, Eastern Hararghe, Ethiopia. RESULTS: Out of 256 samples examined, Pasteurella was isolated in 64 (25%), of which 38 (59.4%) were from lungs and 26 (40.6%) were from nasal cavities. 87.5% of the isolates were Mannheimia haemolytica and 12.5% were Pasteurella multocida. All of the isolates from the lungs were Mannheimia haemolytica whereas 69% of the isolates from nasals cavities were Mannheimia haemolytica. Age and body temperature were significantly associated with Pasteurella isolates from clinic (P < 0.05). Despite diverse in the site of origins, the isolates exhibited uniformity in sensitivity to a majority of the antibacterial agents. The most effective drug was Cholramphenicol (100%) followed by Sulfamethoxazole (89.1%) and Tetracycline (84.4%). Both species were completely resistant to Gentamycin and Vancomycin. CONCLUSION: Mannheimia haemolytica is the most common cause of ovine pneumonic pasteurellosis in the study area. The isolates were susceptible to limited antimicrobial agents. Therefore, the antimicrobial susceptibility test should be conducted before treatment, except for critical cases.

A herbal antifungal formulation of Thymus serpillum, Origanum vulgare and Rosmarinus officinalis for treating ovine dermatophytosis due to Trichophyton mentagrophytes.

A number of herbal products with anti-inflammatory, antiseptic and antimycotic properties are available for dermatological usage. The successful treatment of 13 sheep affected by ringworm due to Trichophyton mentagrophytes with a mixture consisting of essential oils (EOs) of Thymus serpillum 2%, Origanum vulgare 5% and Rosmarinus officinalis 5% in sweet almond (Prunus dulcis) oil. The effectiveness of EOs and of the major components of the mixture (thymol, carvacrol, 1,8 cineole, α-pinene, p-cymene, γ-terpinene) against the fungal clinical isolate was evaluated by a microdilution test. Thirteen animals were topically administered with the mixture twice daily for 15 days. The other sheep were administered with a conventional treatment (seven animals) or left untreated (two animals). Minimum inhibitory concentration (MIC) values were 0.1% for T. serpillum, 0.5% for O. vulgare, 2.5% for I. verum and 5% for both R. officinalis and C. limon. Thymol and carvacrol showed MICs of 0.125% and 0.0625%. A clinical and aetiological cure was obtained at the end of each treatment regimen in only the treated animals. Specific antimycotic drugs licenced for food-producing sheep are not available within the European Community. The mixture tested here appeared to be a versatile tool for limiting fungal growth.

[Infectious causes of lameness in sheep - treatment options in veterinary practice]. / Infektiöse Lahmheiten beim Schaf - Therapiemöglichkeiten in der tierärztlichen Praxis.

Lameness in sheep induced by infectious agents can cause problems regarding animal welfare, diagnosis and treatment. Individual lame animals and lameness caused by systemic disease, but especially flock problems due to infectious causes of lameness, such as foot rot, can for various reasons pose a problem for veterinary treatment. The causes of lameness in sheep are described with a special focus on infectious flock problems, and the different treatment options such as foot trimming, foot baths and local and systemic antibiotic therapy as well as vaccination are discussed based on a review of recent international research. The limited choice of drugs licensed for use in sheep in Germany is highlighted. Treatment, therefore, often requires off-label use or the import of footbathing agents licensed in other European countries. The legal consequences of this lack of nationally licensed veterinary products in dealing with a "minor species" are discussed, with a final call for political solutions that will help improve this unsatisfactory situation.

Vaccination prepartum enhances the beneficial effects of melatonin on the immune response and reduces platelet responsiveness in sheep.

BACKGROUND: Melatonin regulates several physiological processes and its powerful action as antioxidant has been widely reported. Melatonin acts modulating the immune system, showing a protective effect on the cardiovascular system and improving vaccine administration as an adjuvant-like agent. Here, we have investigated the role of melatonin as an adjuvant of the Clostridium perfringens vaccine in prepartum sheep and whether melatonin modulates platelet physiology during peripartum. RESULTS: The experiments were carried out in peripartum sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by ELISA and sheep platelet aggregation was monitored using an aggregometer. Here we demonstrated for the first time that plasma melatonin concentration were higher in pregnant (125 pg/mL) than in non-pregnant sheep (15 pg/mL; P < 0.05). Administration of melatonin prepartum did not significantly modify platelet function but significantly improved the immune response to vaccination against C. perfringens. CONCLUSION: Administration of melatonin as an adjuvant provides a significant improvement in the immune response to vaccine administration prepartum against C. perfringens.

Investigations concerning the prevalence of Coxiella burnetii and Chlamydia abortus in sheep in correlation with management systems and abortion rate in Lower Saxony in 2004.

The intracellular bacteria Coxiella (C) burnetii and Chlamydia (Chl) abortus induce abortion in sheep and also affect humans. While Chl. abortus only infrequently infects humans, C burnetii is the aetiological agent of numerous Q fever outbreaks during the last decades. There is only limited knowledge about the prevalence of both pathogens in sheep, although sheep are involved in almost all Q fever outbreaks in Germany. The aim of our study was to investigate the prevalence of both pathogens in flocks located in Lower Saxony, Germany, in correlation to the management form and abortion rate. Serum samples of 1714 sheep from 95 flocks located in Lower Saxony were investigated by ELISA. 2.7% of these samples were positive, 1.3% showed inconclusive results in the C. burnetii-ELISA. Elevated intra-flock seroprevalences were only detected in three migrating flocks. Chlamydia-specific antibodies could be detected in 15.1% serum samples of mainly shepherded and migrating flocks. In one of these flocks with a high intra-flock seroprevalence for C burnetii (27%) and Chlamydia (44.9%), C burnetii was detected in 21.6% of the placenta samples of normal births and in 12.5% of the colostrum samples by PCR. Aborted fetuses and the corresponding placentas were negative in C burnetii-PCR, but in most of them and also in many other placenta samples Chl. abortus could be detected by PCR and DNA microarray. This survey shows a low overall prevalence of C. burnetii in sheep in Lower Saxony in the year 2004. However, three migrating flocks with a high intra-flock prevalence are localized in the southern parts of Lower Saxony. Spreading of C burnetii could occur, because of the large radius of grazing of all three flocks.

Higher whole-blood selenium is associated with improved immune responses in footrot-affected sheep.

We reported previously that sheep affected with footrot (FR) have lower whole-blood selenium (WB-Se) concentrations and that parenteral Se-supplementation in conjunction with routine control practices accelerates recovery from FR. The purpose of this follow-up study was to investigate the mechanisms by which Se facilitates recovery from FR. Sheep affected with FR (n = 38) were injected monthly for 15 months with either 5 mg Se (FR-Se) or saline (FR-Sal), whereas 19 healthy sheep received no treatment. Adaptive immune function was evaluated after 3 months of Se supplementation by immunizing all sheep with a novel protein, keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity (DTH) skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. Innate immunity was evaluated after 3 months of Se supplementation by measuring intradermal responses to histamine 30 min after injection compared to KLH and saline, and after 15 months of Se supplementation by isolating neutrophils and measuring their bacterial killing ability and relative abundance of mRNA for genes associated with neutrophil migration. Compared to healthy sheep, immune responses to a novel protein were suppressed in FR-affected sheep with smaller decreases in FR-affected sheep that received Se or had WB-Se concentrations above 250 ng/mL at the time of the immune assays. Neutrophil function was suppressed in FR-affected sheep, but was not changed by Se supplementation or WB-Se status. Sheep FR is associated with depressed immune responses to a novel protein, which may be partly restored by improving WB-Se status (> 250 ng/mL).

Antimicrobial resistance and small ruminant veterinary practice.

Antimicrobial resistance (AMR) is recognized as an emerging issue in the practice of veterinary medicine. Although little surveillance and research has been completed on the prevalence of AMR and associated risk factors in small ruminants, evidence of AMR is present in many countries. Furthermore, antimicrobial use (AMU) practices in sheep have been shown to be associated with increased resistance, highlighting the issue of prudent use of these drugs in many countries. Furthermore, AMU practices in sheep have been shown to be associated with increased resistance, highlighting the issue of prudent use of these drugs.

Pharmacodynamics of amoxicillin against Mannheimia haemolytica and Pasteurella multocida and pharmacokinetic/pharmacodynamic (PK/PD) correlation in sheep.

Silicone-made tissue cages were implanted in sheep. Blood serum (SBS) and tissue cage fluid (TCF) samples were collected after amoxicillin intravenous and intramuscular administrations, at the dose of 15 mg/kg. Amoxicillin pharmacodynamics were studied in an artificial culture medium, SBS and TCF with use of a Mannheimia haemolytica and a Pasteurella multocida strain. A concentration-independent antimicrobial activity of amoxicillin was confirmed for levels higher than 0.79-1.75×MIC. This result favored the use of the percentage of the 24 h dosing interval during which drug levels remain above MIC as the appropriate pharmacokinetic/pharmacodynamic index. The subsequent correlation revealed that intravenous administration could be considered effective against "deep" infections caused by bacteria with MICs<1 µg/mL or "shallow" infections caused by bacteria with MICs<0.1 µg/mL. Intramuscular administration could be safely considered effective against both "deep" and "shallow" infections when the MICs of the targeted pathogens are lower than 1 µg/mL.

Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep.

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vaccine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n=14) or used as unvaccinated control (group B, n=8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration.

Effect of parenteral selenium administration to sheep on prevalence and recovery from footrot.

BACKGROUND: Insufficient intake of selenium (Se) is common in many regions, and can contribute to increased susceptibility to and prolonged recovery from infectious diseases. OBJECTIVE: To determine the effect of Se administration in decreasing the severity and prevalence of footrot in sheep. ANIMALS: Thirty-eight footrot-affected and 19 nonaffected sheep from a commercial flock of known high incidence of footrot. METHODS: Placebo-controlled, prospective, 15-month clinical trial. Footrot-affected sheep were randomly assigned into 2 groups (n = 19) and injected with either 5 mg Se (footrot [FR]-Se) or saline (FR-Sal) at 1-month intervals for the duration of the study. Unaffected sheep (controls) received no treatment. Sheep feet were examined, trimmed, and scored for footrot with a scale of 0 (no footrot) to 4 (extensive) at 0, 3, 6, 9, and 15 months. Sheep were also bled at time 0 and then at 3, 6, and 15 months to assess whole blood Se concentrations. RESULTS: At time 0, control sheep (255 +/- 11 ng/mL) had higher (P < .05) whole blood Se concentrations compared with FR-Se (205 +/- 12 ng/mL) and FR-Sal (211 +/- 14 ng/mL) sheep. By 6 months, FR-Se sheep (317 +/- 9 ng/mL) had whole blood Se concentrations greater (P < .05) than both control (281 +/- 14 ng/mL) and FR-Sal (277 +/- 16 ng/mL) sheep. FR-Se ewes showed a faster decline in highest lesion score at 3 (P= .012) and 6 (P= .0036) months, and a greater decrease in the number of feet with foot score >0 at 6 (P= .020) months compared with FR-Sal ewes. Sheep with blood Se concentrations <300 ng/mL were at 3.5 times greater risk (1.1-12.1 confidence interval, odds ratio) for FR, although this relationship was only significant (P= .04) at 6 months of the study. CONCLUSIONS AND CLINICAL IMPORTANCE: In sheep with footrot, improved Se status in conjunction with routine control practices result in more rapid improvement of foot lesions.

Melatonin enhances the immune response to vaccination against A1 and C strains of Dichelobacter nodosus.

Melatonin has been shown to exert immunomodulatory properties with broad application in veterinary medicine. Here we have investigated the effect of exogenous melatonin in the improvement of the immune response to administration of an immune-preparation of two stumps of A1 and C strains of Dichelobacter nodosus in sheep. Subcutaneous administration of melatonin enhanced plasma levels of melatonin from days 42 to 120. Administration of melatonin to vaccinated animals enhanced both the titer of antibodies and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Our results suggest that melatonin increased the immune response to vaccination and open new perspectives in the design of prophylactic strategies.

An oil-emulsion vaccine induces full-protection against Mycoplasma agalactiae infection in sheep.

The immunogenicity and efficacy of three inactivated vaccines (A, B, C) prepared with Mycoplasma agalactiae (M. agalactiae) and with different oil-emulsion adjuvants were evaluated in sheep. Twenty-eight animals were used, divided into four groups (a, b, c, d) of seven animals each. Three groups were immunized with the same vaccine, but using different adjuvants, while one group (d) was used as an unvaccinated control group. All the vaccine formulations were able to induce clinical protection of animals after challenge with M. agalactiae, but only vaccine C, emulsioned with Montanide ISA-563, Marcol-52 and Montane-80 (ratio: 30%, 63%, 7% respectively), was able to induce full protection in challenged animals, preventing both the onset of clinical signs and infection by M. agalactiae.

In vitro antimycoplasmal activity of six Jordanian medicinal plants against three Mycoplasma species.

The in vitro effect of six Jordanian traditional medicine plant methanolic extracts were tested against 32 isolates of Mycoplasma species; Mycoplasma mycoides subsp. mycoides LC (6), Mycoplasma capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions in Jordan. All Mycoplasma species showed susceptibility to Artemisia herba-alba and Artemisia arborescens with MIC ranges from 3.125-12.5 mg/ml. Allium sativum and Punica grantum showed limited activity against some Mycoplasma isolates. Olea europea and Citrullus colocynthis showed no in vitro activity against any of the Mycoplasma species tested. Artemisia herba-alba and Artemisia arborescens may therefore be useful for the treatment of mycoplasma infections.

First serological and molecular evidence on the endemicity of Anaplasma ovis and A. marginale in Hungary.

Recurring and spontaneously curing spring haemoglobinuria was recently reported in a small sheep flock in a selenium deficient area of northern Hungary. In blood smears of two animals showing clinical signs, Anaplasma-like inclusion bodies were seen in erythrocytes. To extend the scope of the study, 156 sheep from 5 flocks and 26 cattle from 9 farms in the region were examined serologically with a competitive ELISA to detect antibodies to Anaplasma marginale, A. centrale and A. ovis. The seropositivity in sheep was 99.4%, and in cattle 80.8%. A. ovis and A. marginale were identified by PCR and sequence analysis of the major surface protein (msp) 4 gene in sheep and cattle, respectively. Haemoglobinuria, an unusual clinical sign for anaplasmosis might have been a consequence of transient intravascular haemolysis facilitated by selenium deficiency in recently infected sheep, as indicated by the reduction of mean corpuscular haemoglobin concentration (MCHC). Membrane damage was also demonstrated for parenchymal cells, since their enzymes showed pronounced elevation in the plasma. Ticks collected from animals in the affected as well as in neighbouring flocks revealed the presence of Dermacentor marginatus, Ixodes ricinus and D. reticulatus, with the dominance of the first. The present data extend the northern latitude in the geographical occurrence of ovine anaplasmosis in Europe and reveal the endemicity of A. ovis and A. marginale in Hungary.