Contagious agalactia is a serious disease of small ruminants affecting mainly mammary glands, joints and eyes. In sheep, the main aetiological agent is Mycoplasma agalactiae (Ma) whose abilities to persist in the target organs are known. Since there is no information on the effect of acute and chronic Ma infection on circulating leucocytes, the present study was designed to monitor granulocytes, monocytes, T and B lymphocytes, by flow cytometry, in female lactating sheep nasally infected with Ma. A profound depletion of leucocytes was observed from day 5 to day 34 post infection (p.i.). In particular, while the granulocytes returned to baseline levels by day 12 p.i., the monocytes remained significantly low until day 20 p.i. The infection caused a prolonged depletion of peripheral T lymphocytes (both CD4(+) and CD8(+)) while B lymphocytes remained unaltered throughout the study. Mycoplasma agalactiae was detected by real-time PCR in several anatomical sites (ear, nose and milk) from day 2-5 p.i. until the end of the study (i.e., day 50 p.i.) while a transient bacteraemia was observed from day 5 to day 12 p.i. The leucopenia observed following intranasal Ma infection is likely due to leucocyte infiltration within the target organs.

A catalase-negative MRSA strain and a methicillin resistant S. pseudintermedius strain (MRSP) were isolated from a dog affected by a severe form of pododermatitis. The catalase negative isolate was typed as a SCCmec I, PVL negative, ST5 t002 strain. A deletion at position 487 of the kat gene altered the functionality of the catalase enzyme. This is the first report of a catalase-negative MRSA in animals. As catalase test is a rapid assay routinely employed for the identification of staphylococci in clinical microbiology laboratories, the presence of MRSA with this uncommon phenotype may be underestimated. Moreover, catalase-negative staphylococci should be investigated more in-depth in order to assess their virulence.

An observational study was conducted to ascertain the clinical manifestation of Contagious Bovine Pleuropneumonia (CBPP) in the Western Province of Zambia following an outbreak in an endemic region. It was observed that both calves and adult cattle in 9 herds that were not vaccinated with CBPP T1/44 vaccine reported mortality while 24 herds that had consistently been vaccinated against CBPP only reported mortalities in calves between 2 months and 8 months of age. This observation indicates that in CBPP endemic areas, calves may acquire the disease from the carriers in the infected herds. It may also indicate that cattle vaccinated with the CBPP T1/44 vaccine, when challenged with the field strain, shed the infective bacteria but may not necessarily develop clinical disease. This observation also suggests that calves may not be protected by maternal antibodies obtained passively through colostrum from the vaccinated dams and thus may succumb to the disease with typical clinical signs similar to those observed in older cattle. The result in this study further suggests that it might be necessary to vaccinate calves a short while after birth to boost their immunity against CBPP. It is therefore necessary to conduct further investigation of CBPP in calves in endemic situations.

An epidemiological survey for canine parvovirus (CPV) and canine coronavirus (CCoV) was 25 conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District 26 of Tirana during 2011-2013. The molecular analyses detected 53 and 31 CPV and CCoV positive 27 specimens, respectively, with mixed CPV/CCoV infections being diagnosed in 28 dogs. The most 28 frequently detected CPV type was CPV-2a, whereas CCoV-IIa was the predominant CCoV subtype. 29 A better comprehension of the CPV/CCoV epidemiology in eastern European countries will help 30 assess the most appropriate vaccination strategies to prevent these widespread agents of canine 31 acute gastroenteritis.

An epidemiological survey for Canine parvovirus 2 (CPV-2) and Canine coronavirus (CCoV) was conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District of Tirana during 2011-2013. The molecular assays detected 53 and 31 CPV- and CCoV-positive specimens, respectively, with mixed CPV-CCoV infections diagnosed in 28 dogs. The most frequently detected CPV type was 2a, whereas IIa was the predominant CCoV subtype. A better comprehension of the CPV-CCoV epidemiology in eastern European countries will help to assess the most appropriate vaccination strategies to prevent disease due to infections with these widespread agents of acute gastroenteritis in the dog.

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.

In the last decade there has been a rapid global spread of methicillin-resistant Staphylococcus pseudintermedius (MRSP) clones displaying multidrug resistance in dogs. We investigated prevalence, antimicrobial susceptibility and clonal distribution of MRSP isolated from clinical canine samples between during 2011-2014. Following species identification by nuc PCR, MRSP were confirmed by the presence of mecA and characterized by antimicrobial susceptibility testing, Pulsed Field Gel Electrophoresis (PFGE), SCCmec typing, and Multi-Locus Sequence Typing (MLST) of a few isolates having distinct PFGE profiles. Both the MRSP isolation frequency in the 175 samples tested (12%) and the prevalence of methicillin resistance amongst the 63S. pseudintermedius isolates (33%) were high compared to a previous study in Italy. Sequence type (ST)71 carrying SCCmec type II-III, described as the epidemic European MRSP clone, accounted for approximately half of the isolates. The remaining isolates belonged to ST410-SCCmec type II-III, ST258-SCCmec type IV and other three clones associated with SCCmec type IV (ST261, ST290 and ST477). MRSP were consistently resistant to potentiated sulfonamides, and more frequently to clindamycin, ciprofloxacin and doxycycline than methicillin-susceptible isolates. Gentamicin was the only antibiotic showing good in vitro activity on all MRSP with 20 of the 21 isolates being susceptible. Results confirm a high prevalence of MRSP amongst clinical samples in Italy, revealing the emergence of new clones other than ST71, such as ST258, ST410, ST261, ST290 and ST477, here describe for the first time. Implementation of antimicrobial stewardship and surveillance programmes are required to prevent the emergence of new MRSP clones and reducing transmission in small animal practice.

BACKGROUND: In this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil.The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field investigation. RESULTS: The results indicated that, in contrast to the classic method, the GABRI method was able to detect B.anthracis in all contaminated samples. The GABRI method produces a more sensitive measure of anthrax spore presence significantly different from the standard method. In particular, the latter is more sensitive to the presence of normal soil contaminants. CONCLUSION: The main feature of the GABRI method is its ability to strongly reduce the presence of the environmental contaminants which being much more numerous than B.anthracis tend to inhibit their germination and growth making it extremely difficult to visualize any colonies. The reduction of the microbial environment also allows one to be able to culture and test a larger quantity of potentially contaminated soil and to isolate B. anthracis when the spores are present in very low concentrations in the soil.

Salmonella enterica subsp. enterica serovar Abortusequi is frequently reported as a cause of abortion in mares and neonatal septicemia and polyarthritis in Asian and African countries, but only sporadically in Europe and the United States. We report an outbreak of S. Abortusequi in foals in Italy, characterized by high mortality. In a herd of Murgese horses, 10 of 34 newborns died at birth and a further 7 died, after developing severe clinical signs, during the first 10 d of life. Tissue specimens from different organs of 2 dead foals, synovial fluids from 4 sick foals, and vaginal and rectal swabs from their dams were cultured. A total of 16 isolates, all as pure cultures, were obtained and identified as Salmonella. The isolates exhibited the same antimicrobial resistance pattern and the same sequence type, ST251, a type that has been associated with S. Abortusequi. Six of 16 isolates were serotyped and found to be S. Abortusequi 4,12:-:e,n,x. Equine practitioners should be aware of S. Abortusequi infection as a cause of neonatal mortality in foals.

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vac- cine 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.

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 100-101 viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.

Contagious bovine pleuropneumonia (CBPP) is a disease of economic importance that is widely distributed in sub-Saharan African and contributes significantly to cattle morbidity and mortality. Lack of resources to implement eradication measures has led to the disease becoming endemic in most areas in sub-Saharan Africa where governments have little resources and the majority of the people are poor. Usually, control and eradication of such diseases as CBPP is treated as a public good by governments and to achieve this, governments are usually assisted by nongovernment organisations, bilateral government programmes and international donors. The private sector, which usually is companies that run businesses to make profit, although not very well established in sub-Saharan Africa could play a big role in the eradication of CBPP in the region. This could play a dual role of promoting investment and also eradicate livestock diseases which have proved a menace in the livestock sector. This paper highlights the role played by the private sector in the control of CBPP in Zambia.

Contagious bovine pleuropneumonia (CBPP), a highly infectious and fatal disease of cattle present in many countries in sub-Saharan Africa, is usually controlled by mass vaccinations.However, vaccination against CBPP is known to cause site reactions in a percentage of cattle especially in primary vaccinations. In Zambia, a record of site reactions was kept for seven consecutive years from 2005 to 2011 to establish the level of the problem. In some areas, after 3 years of consecutive vaccination campaigns, immunization could not be implemented for a period of 2 years because of logistical difficulties or owner resistance. Whereas in the three preceding years when animals were vaccinated annually, site reactions were in the range of 6.2 %; on resumption of vaccination in the herds that had not been immunized for 2 years, site reactions averaged 21.3 %. This data shows that the T1/44 vaccine may cause severe local reactions in cattle if there is any break in annual vaccinations. It is therefore important for authorities to ensure that the cattle at risk of contracting CBPP are regularly vaccinated to avoid discouraging farmers from presenting their animals.

Bovine brucellosis is diagnosed by official tests, such as Rose Bengal plate test (RBPT) and Complement Fixation test (CFT). Both tests detect antibodies directed against the lipolysaccharide (LPS) of Brucella cell wall. Despite their good sensitivity, those tests do not discriminate between true positive and false positive serological reactions (FPSR), the latter being generated by animals infected with other Gram negative microorganisms that share components of Brucella LPS. In this study, an antigenic extract from whole Brucella melitensis B115 strain was used to set up an ELISA assay for the serological diagnosis of bovine brucellosis. A total of 148 serum samples from five different groups of animals were tested: Group A: 28 samples from two calves experimentally infected with Yersinia enterocolitica O:9; Group B: 30 samples from bovines infected with B. abortus; Group C: 50 samples from brucellosis-free herds; Group D: 20 samples RBPT positive and CFT negative; Group E: 20 samples both RBPT and CFT positive. Group D and Group E serum samples were from brucellosis-free herds. Positive reactions were detected only by RBPT and CFT in calves immunized with Y. enterocolitica O:9. Sera from group B animals tested positive also in the ELISA assay, whereas sera from the remaining groups were all negative. The results obtained encourage the use of the ELISA assay to implement the serological diagnosis of brucellosis.