Mastitis In Sheep Research Articles

Divergence of a strain of Pseudomonas aeruginosa during an outbreak of ovine mastitis

Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections.

Genetic Parameters for Somatic Cell Count, Logscc and Somatic Cell Score of Breeds: Improved Valachian, Tsigai, Lacaune and their Crosses/Genetski Parametri Za Broj Somatskih Ćelija, Logscc I Skor Somatskih Ćelija Kod Rasa: Poboljšana Vlaška, Cigaja, Lakon I Njihovih Meleza

Abstract In the last few years there has been increasing emphasis on reducing milk somatic cell count to improve the milk quality in dairy ruminants. Genetic parameters for somatic cell count (SCC), LOGSCC and somatic cell score (SCS) were estimated. About 1193 measurements were included in the analysis for each character of 358 ewes of 9 genotypes. Nine breeds and genotypes were included in these experiments: purebred Improved Valachian (IV), Tsigai (T), Lacaune (LC) ewes, and IV and T crosses with a genetic portion of Lacaune and East Friesian (EF) - 25 %, 50 % and 75 %. Primary data were processed using restricted maximum likelihood (REML) methodology and the multi-trait animal model, using programs REMLF90 and VCE 4.0. Heritability coefficients for somatic cell count were low: h2=0.03, for LOGSCC h2= 0.08 and for somatic cell score h2=0.06. Somatic cell score can be considered for inclusion in a breeding program aimed at reducing somatic cell count and frequency of clinical mastitis in dairy sheep.

Sequence diversity, cytotoxicity and antigenic similarities of the leukotoxin of isolates of Mannheimia species from mastitis in domestic sheep

Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles.

Survey of accessory gene regulator (agr) groups and TSST-1 encoding gene (tst) in Staphylococcus aureus isolated from ewes with mastitis in the northwest of Iran

Summary Accessory gene regulator (agr locus) is a global regulator of many virulence gene expressions in Staphylococcus aureus. Four interference classes related to genetic polymorphisms in the agr locus have so far been described. In the present study, the agr locus specificity groups were studied within a total of 43 S. aureus isolates which were isolated from ewes with mastitis in three regions in the Northwest of Iran. In addition, the isolates were examined for the presence of TSST-1 (toxic shock syndrome toxin-1) encoding gene (tst) using PCR. Identification of agr groups, using agr group-specific multiplex PCR, classified the majority of isolates into group I (51.16%) and to a lesser extent into agr group IV (44.19%). Only one isolate (2.23%) for each of the agr groups II and III was found. This study also indicated that 44.19% of isolates (n=19) possessed the tst gene and that 94.74% (n=18) belonged to agr group I. According to the results, certain agr groups comprised the great majority of sheep mastitis associated S. aureus isolates. Moreover, the high proportion of S. aureus isolates was tst positive, indicating an issue requiring consideration as it is relevant to food hygiene. The results also showed a variation in distribution of the tst gene among the different agr groups.

Deletion variant near ZNF389 is associated with control of ovine lentivirus in multiple sheep flocks

Ovine lentivirus (OvLV) is a macrophage-tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker-assisted selective breeding might improve odds of infection and control of OvLV post-infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post-infection in multiple animal sets. Proviral concentration is a live-animal diagnostic measure of OvLV control post-infection related to severity of OvLV-induced lesions. A recent genome-wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/μg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV-positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post-infection in multiple sheep flocks.

Trematode infections in pregnant ewes can predispose to mastitis during the subsequent lactation period

Objective was to investigate if trematode infections predispose ewes to mastitis and/or metritis. We used 80 trematode-infected ewes: primigravidae in group P-A and multigravidae in M-A remained untreated, primigravidae in P-B and multigravidae in M-B were drenched with netobimin and multigravidae in M-C were given rafoxanide. We collected faecal samples for parasitological examination, blood samples for β-hydroxybutyrate concentration measurement and uterine content, teat duct material and milk samples for bacteriological examination. We found significant differences in blood β-hydroxybutyrate concentrations between M-A, M-B and M-C during pregnancy (P⩽0.002). We did not observe significant differences between groups regarding development of metritis (P>0.83). We found that for M-A, M-B and M-C ewes, respectively, median time to first case of mastitis was 5.75, 21 and 6.75days after lambing (P=0.003) and incidence risk of mastitis was 0.308, 0.069 and 0.222 (P=0.047). We postulate that trematode infections predispose ewes to mastitis; perhaps, increased β-hydroxybutyrate blood concentrations adversely affect mammary cellular defences. This is the first report associating parasitic infections with mastitis in sheep.

Detection of Visna Maedi virus in mesenteric lymph nodes and in other lymphoid tissues of sheep three years after respiratory infection

Visna/Maedi virus (VMV), a small ruminant lentivirus responsible for lymphoproliferative pneumonia, encephalitis, arthritis and/or mastitis in sheep, has been detected in different non-lymphoid organs. However, only a few investigations have been carried out in lymphoid tissues. In this study, some lymphoid tissues and lymph node draining or non-draining VMV target organs from five sheep infected experimentally by the respiratory route three years previously were investigated. Archival samples of spleen, red bone marrow, caudal mediastinal lymph nodes, mammary lymph nodes, popliteal lymph nodes and mesenteric lymph nodes were tested by PCR for the presence of proviral DNA. Popliteal and mesenteric lymph node samples were tested also by immunohistochemical staining of the viral capsid antigen p28. The proviral DNA was detected by PCR in all the lymphoid tissue samples from the infected sheep. The viral antigen was stained in mononuclear cells in popliteal and mesenteric lymph nodes of the infected sheep. Although the lymph nodes draining the classical target organs seem to be more infected than the others, both the viral capsid antigen and the proviral DNA were present also in lymph nodes draining non-target organs, such as the mesenteric lymph nodes. These findings show the presence of VMV in different lymphoid tissues in the late stages of infection and suggest a potential role of these tissues as a site for viral reservoir and replication, even three years after infection. &amp;nbsp;

Comparative molecular analysis of ovine and bovine Streptococcus uberis isolates

Streptococcus uberis causes clinical and subclinical mastitis in cattle and sheep, but it is unknown whether the composition of Strep. uberis populations differs between host species. To address this, we characterized a collection of bovine and ovine Strep. uberis isolates with shared geographical and temporal origins by means of an expanded multilocus sequence typing scheme. Among 14 ovine and 35 bovine isolates, 35 allelic profiles were detected. Each allelic profile was associated with a single host species and all but one were new to the multilocus sequence typing database. The median number of new alleles per isolate was higher for ovine isolates than for bovine isolates. None of the ovine isolates belonged to the global clonal complexes 5 or 143, which are commonly associated with bovine mastitis and which have a wide geographical distribution. Ovine isolates also differed from bovine isolates in carriage of plasminogen activator genes, with significantly higher prevalence of pauB in ovine isolates. Isolates that were negative for yqiL, one of the targets of multilocus sequence typing, were found among ovine and bovine isolates and were not associated with a specific sequence type or global clonal complex. One bovine isolate carried a gapC allele that was probably acquired through lateral gene transfer, most likely from Streptococcus salivarius. We conclude that ovine isolates are distinct from bovine isolates of Strep. uberis, and that recombination between isolates from different host species or bacterial species could contribute to changes in virulence gene profiles with relevance for vaccine development.

Mastitis detection in sheep by infrared thermography

This study aims to evaluate the use of an infrared thermograph for mastitis diagnosis in sheep. Thirty-seven Santa Inês ewes were evaluated weekly through infrared images obtained with thermograph FLIR System Series-i®. Milk was collected for somatic cell count and milk compound level determination. The clinical mastitis group had the highest fat and protein level, as well as the lowest lactose level. The udder temperatures were higher for subclinical mastitis group. The udder temperature data was able to correctly classify the animals into the mastitis groups and the canonical analysis showed that these temperatures clearly differentiated the subclinical mastitis groups from the others. Therefore, this study showed that udder infrared temperatures can be used as diagnostic method to mastitis in sheep.

Tissue distribution of enrofloxacin after intramammary or simulated systemic administration in isolated perfused sheep udders

To determine the tissue distribution of enrofloxacin after intramammary or simulated systemic administration in isolated perfused sheep udders by measuring its concentration at various sample collection sites. 26 udders (obtained following euthanasia) from 26 healthy lactating sheep. For each isolated udder, 1 mammary gland was perfused with warmed, gassed Tyrode solution. Enrofloxacin (1 g of enrofloxacin/5 g of ointment) was administered into the perfused gland via the intramammary route or systemically via the perfusion fluid (equivalent to a dose of 5 mg/kg). Samples of the perfusate were obtained every 30 minutes for 180 minutes; glandular tissue samples were obtained at 2, 4, 6, and 8 cm from the teat base after 180 minutes. The enrofloxacin content of the perfusate and tissue samples was analyzed via high-performance liquid chromatography with UV detection. After intramammary administration, maximun perfusate enrofloxacin concentration was detected at 180 minutes and, at this time, mean tissue enrofloxacin concentration was detected and mean tissue enrofloxacin concentration was 123.80, 54.48, 36.72, and 26.42 μg/g of tissue at 2, 4, 6, and 8 cm from the teat base, respectively. Following systemic administration, perfusate enrofloxacin concentration decreased with time and, at 180 minutes, tissue enrofloxacin concentrations ranged from 40.38 to 35.58 μg/g of tissue. By 180 minutes after administration via the intramammary or systemic route in isolated perfused sheep mammary glands, mean tissue concentration of enrofloxacin was greater than the minimum inhibitory concentration required to inhibit growth of 90% of many common mastitis pathogens in sheep. Use of either route of administration (or in combination) appears suitable for the treatment of acute mastitis in sheep.

Molecular epidemiology ofMannheimia haemolyticaandMannheimia glucosidaassociated with ovine mastitis

While Mannheimia haemolytica and Mannheimia glucosida have been recognized as causes of intramammary infection in sheep, there has been no investigation of the epidemiology of the strains involved. Pulsed field gel electrophoresis was used to study the molecular epidemiology of isolates of these 2 species associated with ovine mastitis. Ten distinct strains were recognized among 12 M. haemolytica isolates, and 7 distinct strains among 13 M. glucosida isolates. The results demonstrate a high diversity of isolates with the ability to cause ovine mastitis. However, the presence of some identical isolates may suggest the possibility of horizontal transmission of these species in some flocks, possibly through lamb sucking, and/or differences in the capacity of some isolates to cause mastitis in sheep.

Outbreak of mastitis in sheep caused by multi-drug resistantEnterococcus faecalisin Sardinia, Italy

An outbreak of infective mastitis due to Enterococcus faecalis occurred in an intensive sheep farm in north Sardinia (Italy). E. faecalis, which is only rarely isolated from sheep milk, was unexpectedly found in 22·3% of positive samples at microbiological examination. Forty-five out of the 48 E. faecalis isolates showed the same multi-drug resistance pattern (cloxacillin, streptomycin, kanamycin, clindamycin, oxytetracycline). E. faecalis isolates were analysed by pulsed-field gel electrophoresis, and all 45 multi-drug resistant strains showed an indistinguishable macrorestiction profile, indicating their clonal origin. To our knowledge, this is the first report of an outbreak of mastitis in sheep caused by E. faecalis.

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria.

Koyunlarda Gangrenöz Mastitise Neden Olan Bakterilerin İdentifikayonu ve Gangrenli Sütlerdeki Staphylococcus aureus’un PCR ile Tespit Edilmesi

Summary The objective of this study was to identify bacteria causing gangrenous ovine mastitis as well as to detect and genotype Staphylococcus aureus in milk samples by using polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism (RFLP) of coagulase gene. A total of 264 milk samples were collected from Awassi sheep with gangrenous mastitis raised in Sanliurfa province of Turkey. By using bacteriological examination Staphylococcus aureus (75%), Mannheimia haemolytica (7.2%), coagulase negative staphylococci (6.43%), Escherichia coli (4.55%), Corynebacterium spp. (0.76%) and Actinomyces pyogenes (0.76%) were identified. Bacterial DNA was isolated from 155 randomly selected milk sample. By PCR amplification of coagulase gene S. aureus was detected in 91.6% of the milk samples. Three different amplicons of 420 (0.7%), 580 (65.3%) and 780 (34%) bp band sizes, each showing a specific RFLP profile were observed. In conclusion, S. aureus was found to be mostly responsible for gangrenous mastitis in sheep raised in this region. S. aureus in milk samples could be more rapidly detected by PCR method. Two coa genotype of S. aureus were found to be common suggesting that limited number of strains were responsible for most cases of gangrenous mastitis in the region under the study.

Subklinik Koyun Mastitislerinden İzole Edilen Koagulaz Negatif Stafilokok Suşlarının Antibiyotik Duyarlılıkları, Direnç Genlerinin Varlığı ve Biyofilm Sentezi

Subklinik Koyun Mastitislerinden İzole Edilen Koagulaz Negatif Stafilokok Suşlarının Antibiyotik Duyarlılıkları, Direnç Genlerinin Varlığı ve Biyofilm Sentezi

Mastitis in Small Ruminants

This paper reviews the epidemiology, etiologies, risk factors, and preventive management strategies used to minimize mastitis in dairy sheep and dairy goats. Clinical mastitis typically occurs in &lt;5% of lactating does and ewes, but subclinical mastitis may occur in up to 15-30% of animals. Somatic cell counts (SCC) of milking ewes can be used to define subclinical mastitis, and a threshold of about 200,000 to 400,000 cells per mL will accurately identify most infected ewes. Interpretation of SCC values of milking goats is complicated by the presence of cytoplasmic particles in milk. However, intramammary infection (IMI) in milking does results in increased SCC values which must be interpreted based on intervening physiological factors such as stage of lactation, parity, and estrus. Milking management and dry-off treatment are important strategies for producers to adopt to minimize the development of new IMI.

The first case of mastitis in sheep caused by Actinobacillus ureae from Iran

Actinobacillus ureae, previously known as Pasteurella urea, is a commensal bacterium, which rarely causes disease in humans and has not been reported from any animal case in the veterinary literature. In this report, the occurrence of acute mastitis caused by A. ureae in an Iranian Ghezel sheep is described.

The Importance of Staphylococci and Threshold Value of Somatic Cell Count for Diagnosis of Sub‐clinical Mastitis in Pirlak Sheep at Mid‐lactation

This study investigated the bacterial agents causing sub-clinical mastitis and the mean somatic cell counts (SCC) of milk in Pirlak sheep at mid-lactation. The percentage of infected udder halves was 11.4% (53/464). The most frequently isolated species were coagulase-negative staphylococci (CNS) (64.2%), followed by Staphylococcus aureus (24.5%) and Escherichia coli (11.3%). Among the CNS, the most common species was Staphylococcus epidermidis (38.2%). The other species isolated from milk samples were Staphylococcus xylosus (17.7%), Staphylococcus chromogenes (14.7%), Staphylococcus simulans (8.8%) and Staphylococcus hyicus (8.8%). The mean SCC for culture positive and negative samples was 1742×10(3) and 161×10(3) cells/ml, respectively. A significant difference (p<0.05) was determined between with and without microbial growth groups in terms of the SCC values. Threshold limit for SCC was 374×10(3) cells/ml for Pirlak sheep. In conclusion, it was considered that SCC is an important predictor of sub-clinical mastitis in Pirlak sheep. This is the first study to describe the bacterial agents causing sub-clinical mastitis and threshold limit for SCC in Pirlak sheep in Turkey.

Etiologia e perfil de sensibilidade de bactérias isoladas de ovelhas com mastite na região nordeste do estado do Pará

Objetivou-se com este trabalho estudar a etiologia da mastite em ovelhas na região nordeste do Pará, além de estabelecer o perfil de sensibilidade das bactérias isoladas frente a antimicrobianos. Foram examinadas 176 ovelhas da raça Santa Inês, em lactação, mantidas em sistema semi-intensivo, pertencentes a sete propriedades especializadas na criação de ovinos. Foi realizado o exame clínico da glândula mamária, o exame macroscópico da secreção láctea por meio do Teste da Caneca Telada, o California Mastitis Test (CMT), o exame microbiológico do leite e o antibiograma. Das 352 metades mamárias estudadas (176 ovelhas), 21 (5,97%) apresentaram mastite clínica, 26 (7,39%) apresentaram mastite subclínica e 305 (86,64%) metades mamárias foram negativas. A maioria dos animais acometidos pela mastite estava no terço médio da lactação, com menor número de crias e maior número de lactações. Na mastite clínica (MC) as bactérias isoladas foram Staphylococcus spp. coagulase negativo (42,9%); Staphylococcus aureus (9,52%); Streptococcus spp. (4,76%) e Escherichia coli (4,76%). As associações observadas foram Staphylococcus aureus e Streptococcus spp. (4,76%); Staphylococcus spp. coagulase negativo não hemolítica, Staphylococcus spp. coagulase negativo hemolítica e Staphylococcus spp. coagulase negativo pigmento não hemolítica (4,76%). Já na mastite subclínica (MSC), as bactérias isoladas foram Staphylococcus spp. coagulase negativo (26,9%); Staphylococcus aureus (15,4%); Streptococcus spp. (7,69%); Escherichia coli (7,69%) e Citrobacter freundii (11,5%). A associação observada foi Staphylococcus spp. coagulase negativo não hemolítica e Staphylococcus spp. coagulase negativo hemolítica (3,85%). Os antimicrobianos com maior eficácia contra os agentes isolados Gram positivos foram penicilina/novobiocina (100%), cefalotina (100%) e florfenicol (100%) e contra o Citrobacter freundii foram a ampicilina (100%) e florfenicol (100%). Já em relação a Escherichia coli, 66,7% dos isolados mostraram-se resistentes à ampicilina, cefalotina, florfenicol e tetraciclina. A mastite está presente em ovelhas no estado do Pará, havendo a necessidade de estimar, em estudos futuros, as perdas econômicas causadas por essa enfermidade. O CMT apresentou resultados satisfatórios, podendo ser recomendado como teste de triagem para o diagnóstico de casos individuais de mastite subclínica em ovinos, uma vez que apresentou boa relação com o exame microbiológico. No antibiograma foi observado que a maioria dos agentes isolados apresenta-se sensível aos diferentes antimicrobianos testados, sendo os antibióticos com melhor eficiência o florfenicol e a cefoxitina.

Selenium, vitamin E and vitamin A blood concentrations in dairy sheep flocks with increased or low clinical mastitis incidence

Objective of the study was to evaluate the relationship between blood concentrations of selenium, vitamin E and vitamin A with the incidence of acute clinical mastitis in dairy sheep. For this purpose, 13 flocks with increased (≥10%) and five flocks with small (<3%) incidence rate of clinical mastitis, were studied. In flocks with increased incidence rate, whole blood selenium and serum vitamin A blood concentrations were smaller ( P < 0.001) compared with those in flocks with smaller incidence of the disease; no relationship between vitamin E blood concentration and mastitis incidence was found. Animals with mastitis associated with Staphylococcus aureus had smaller ( P < 0.05) selenium, vitamin E and vitamin A blood/serum concentrations compared to healthy animals within the same flock; animals with mastitis associated with Mycoplasma agalactiae had smaller ( P < 0.05) concentrations of selenium and vitamin A compared to healthy animals within the same flock ( P < 0.05); finally, animals with mastitis associated with coagulase-negative staphylococci had smaller ( P < 0.05) concentrations of selenium compared to healthy animals within the same flock. The results indicate a possible predisposing role of Se deficiency in ovine mastitis. Selenium status of ewes may possibly be used to indicate animals at risk to develop clinical mastitis. Reduced vitamin A serum concentration may also contribute to development of clinical mastitis.