Júlio Carvalheira

Bovine Mastitis Associated with Prototheca blaschkeae

ABSTRACT Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.

E‐cadherin, β‐catenin, invasion and lymph node metastases in canine malignant mammary tumours

Recent studies of canine malignant mammary tumours suggest that reduction of E‐cadherin and/or β‐catenin correlates with invasive behaviour and lymph node metastasis. The aims of this study were to examine the interrelationships between the expression of E‐cadherin and β‐catenin, and the relationship between the expression of E‐cadherin and/or β‐catenin and the mode of growth and metastatic capacity of canine malignant mammary tumours. 90 spontaneous malignant tumours and local and regional lymph nodes were studied. A significant relationship was evidenced between membranous expression of E‐cadherin and β‐catenin (p=0.0027), but not between E‐cadherin and cytoplasmic β‐catenin. Only E‐cadherin as a separate factor was significantly related to tumour invasion (p=0.0072) and lymph node metastasis (p=0.0001). Neither membranous nor cytoplasmic β‐catenin expression was significantly related to either of these phenomena.

Sialyl Lewis x expression in canine malignant mammary tumours: correlation with clinicopathological features and E-Cadherin expression

BackgroundSialyl Lewis x (sLex) antigen is a carbohydrate antigen that is considered not only a marker for cancer but also implicated functionally in the malignant behaviour of cancer cells. Overexpression of sLex is associated with enhanced progression and metastases of many types of cancer including those of the mammary gland. Canine mammary tumours can invade and give rise to metastases via either lymphatic or blood vessels.E-Cadherin is specifically involved in epithelial cell-to-cell adhesion. In cancer, E-Cadherin underexpression is one of the alterations that characterizes the invasive phenotype and is considered an invasion/tumour suppressor gene. Partial or complete loss of E-Cadherin expression correlates with poor prognosis in canine malignant mammary cancer.The aim of this study was to analyse the sLex expression in canine malignant mammary tumours and to evaluate if the presence of sLex correlates with the expression of E-Cadherin and with clinicopathological features.MethodsFifty-three cases of canine mammary carcinomas were analysed immunohistochemically using monoclonal antibodies against sLex (IgM) and E-Cadherin (IgG). The clinicopathological data were then assessed to determine whether there was a correlation with sLex tumour expression. Double labelled immunofluorescence staining was performed to analyse the combined expression of sLex and E-Cadherin.ResultssLex expression was consistently demonstrated in all cases of canine mammary carcinomas with different levels of expression. We found a significant relationship between the levels of sLex expression and the presence of lymph node metastases. We also demonstrated that when E-Cadherin expression was increased sLex was reduced and vice-versa. The combined analysis of both adhesion molecules revealed an inverse relationship.ConclusionIn the present study we demonstrate the importance of sLex in the malignant phenotype of canine malignant mammary tumours. Our results support the use of sLex as a prognostic tumour marker in canine mammary carcinomas. Furthermore, we showed that sLex and E-Cadherin expression were inversely correlated. Future studies are warranted to clarify the molecular mechanism underlying the relation between sLex and E-Cadherin in canine mammary carcinoma cells which represents an important comparative model to woman breast cancer.

Canine parvovirus 2c infection in central Portugal

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.

Immunohistochemical study of the expression of E-cadherin in canine mammary tumours

To investigate the possible role of E-cadherin in canine mammary tumours 20 benign and 40 malignant tumours were evaluated by immunohistochemistry in formalin-fixed paraffin wax-embedded samples. In all the benign tumours, E-cadherin was strongly expressed at the intercellular borders of epithelial cells, but it was less strongly expressed in 17 (43 per cent) of the malignant tumours. Furthermore, poorly differentiated carcinomas were less immunoreactive for E-cadherin than moderately and well differentiated carcinomas.

Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis.

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpsons index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.

Fertility Time Trends in Dairy Herds in Northern Portugal

The economics of dairy production are in great part dictated by the reproductive efficiency of the herds. Many studies have reported a widespread decrease in fertility of dairy cows. In a previous work (Rocha et al. 2001), we found a very poor oestrus detection rate (38%), and consequently a delayed calving to 1st AI and calving to conception intervals. However, a good conception rate at 1st AI was noted (51%) resulting in a low number of inseminations per pregnancy (IAP) (1.4). Here, results from a subsequent fertility time trend assessment study carried out in the same region for cows born from 1992 to 2002 are reported. Statistical linear models were used to analyse the data. Estimate linear contrasts of least square means were computed from each model. The number of observations per studied index varied from 12,130 (culling rate) to 57,589 (non-return rate). Mean age at first calving was 28.9 ± 0.14 months, without (p > 0.05) variation over time. There was a small, but significant (p < 0.05), deterioration of all other parameters. Non-return rates at 90 days and calving rate at 1st AI decreased 0.3% per trimester, with a consequent increase of 0.04 IA/parturition. Oestrus detection rate decreased 0.13% per year, and calving at 1st AI and calving-conception intervals increased 0.17 and 0.07 days/year respectively, while intercalving interval increased 1.7 days per year. From 12,130 cows calving, only 1,816 had a 4th lactation (85% culling/losses). The data was not meant to draw conclusions on the causes for the decreased fertility over time, but an increase of milk production from 6537 kg to 8590 kg (305 days) from 1996 to 2002 is probably one factor to take into consideration. Specific measures to revert or slow down this trend of decreasing fertility are warranted. Available strategies are discussed.

Algaemia in a dairy cow by Prototheca blaschkeae

We describe the first known case of an algaemia by Prototheca blaschkeae in a dairy cow, which occurred after a chronic episode of mastitis caused by this pathogen. The organism was isolated from milk, joint fluid and blood samples, and microbiologic and molecular methods were performed to obtain a definitive identification of the algae. The affected cow was culled only after confirmation of a systemic infection by Prototheca.

Detection and quantitative analysis of human herpesvirus in pilocytic astrocytoma.

We investigated the hypothetical role of human herpesviruses (HHVs) in tumour formation of the cerebellum. Thirty-five samples of pilocytic astrocytoma and 10 control samples of cerebellum from patients who died of unrelated diseases were examined. Presence of the 8 known HHVs was first studied using specific real-time quantitative Polymerase Chain Reaction (qPCR) targeting viral DNA polymerase. HHVs DNA polymerase was found present in 20 samples (7 controls, 13 astrocytomas) and was absent in 25 samples (3 controls, 22 astrocytomas). DNA polymerase of Epstein-Barr Virus (EBV) was present in 16 samples, 7/10 controls (70%) and 9/35 astrocytomas (26%). HHV-1 and Varicella-Zoster virus were detected only twice and HHV-2, Cytomegalovirus, HHV-7 and HHV-8, only once. HHV-6 was not detected. In all cases, the gene copy numbers of DNA polymerase were low (<100/100 ng DNA). A second approach was to search for novel HHVs, using consensus-degenerated hybrid oligonucleotide primers (CODEHOP) PCR: no sequence indicative of a new HHV was detected. In summary, EBV was the most frequent HHV detected in pilocytic astrocytoma, but at very low levels. According to the actually accepted threshold the results suggest that EBV cannot be considered responsible for tumorigenesis of pilocytic astrocytoma.

Natural Coinfection with 2 Parvovirus Variants in Dog

To the Editor: Canine parvovirus (CPV) emerged in the late 1970s, presumably by mutations in feline panleukopenia virus, and became a major viral pathogen of dogs worldwide (1). Between 1979 and 1981, the original type 2 virus (CPV-2) was replaced by a new genetic and antigenic variant, type 2a (CPV-2a). Between 1983 and 1984, CPV-2a was replaced by type 2b (CPV-2b), which differs from type 2a by only 1 epitope located at residue 426 of the VP2 capsid protein (2). CPV-2 does not replicate in cats, but the new variants replicate in dogs and cats (3). Recently, an antigenic change has been observed in a new strain, CPV-2c, isolated from domestic dogs in Italy (4). This variant was also detected in Vietnam (5), other countries in Europe (6), and the United States (7). CPV-2c was recently detected in cats (8) and is characterized by a replacement of aspartic acid with glutamic acid at residue 426 of the VP2 capsid protein. To identify CPV types 2, 2a, and 2b, PCR methods were developed (9). However, these methods could not distinguish type 2c from type 2b (4). Consequently, we used a PCR–restriction fragment-length polymorphism (RFLP) assay with endonuclease MboII. This enzyme can distinguish type 2c from other CPVs (4). Recently, a real-time PCR assay based on minor groove binder (MGB) probe technology was developed for rapid identification and characterization of the antigenic variants. This assay is based on 1 nucleotide polymorphism in the VP2 gene (10). In June 2006, a 10-week-old female dog (PT-32/07) was brought to the veterinary clinic in Figueira da Foz, Portugal, with clinical signs of parvovirus infection, after an episode of gastrointestinal disease in her littermates. Three littermates, also brought to the clinic, showed no signs of infection. None of the dogs were vaccinated against CPV. Clinical signs in dog PT-32/07 were lethargy, anorexia, vomiting, diarrhea, and a temperature of 39.3°C. Identical signs were observed in 1 littermate 3 days later; the 2 other dogs did not show any signs other than lethargy and loose stools. Rectal swab samples from all dogs were screened for CPV by using an immunomigration rapid test (Synbiotics Corporation, Lyon, France). Two of the dogs showed negative results, and 2 showed positive results. Feces, serum, and lingual swab samples were positive for parvovirus DNA. DNA was quantified by using a real-time PCR with TaqMan technology performed in an i-Cycler iQ (Bio-Rad Laboratories, Milan, Italy). CPV variants were characterized by using MGB probe technology. This technology uses type-specific probes labeled with different fluorophores (FAM and VIC) that can detect single nucleotide polymorphisms between CPV types 2a/2b and 2b/2c (10). MGB probes specific for type 2b were labeled with FAM in both type 2a/2b and 2b/2c assays, and MGB probes specific for type 2a (type 2a/2b assay) and type 2c (type 2b/2c assay) were labeled with VIC. All specimens from 1 dog (PT-32/07) were positive for the 2 variants of CPV type 2 (CPV 2b and CPV 2c). Conversely, of the 3 littermates, 2 were positive for CPV type 2b and 1 was positive for CPV type 2c in all samples (Table). Table Detection by minor groove binder probe assay of CPV antigenic variants in different specimens from dogs from the same litter (10 weeks old), Portugal, 2006* A conventional PCR and RFLP analyses were performed by using the method of Buonavoglia et al. (4) with known positive CPV-2b and CPV-2c samples as controls to confirm our findings. The 583-bp PCR product obtained from the coinfected dog by using primer pair 555for/555rev was digested with MboII. Digestion generated 2 fragments (≈500 and 80 bp) in all dog samples. The CPV-2c control sample showed 2 fragments (≈500 and 80 bp), and CPV-2b control sample was not digested with MboII. We report CPV-2b and CPV-2c variants in samples from a dog with littermates that were positive for CPV-2b or CPV-2c during an episode of gastrointestinal disease. Coinfection with multiple CPV variants that showed high genetic diversity in the VP2 gene has recently been reported in a domestic cat (8). Continuous and rapid evolution of CPV may cause serious problems in diagnostic testing and vaccine efficacy. Antigenic variation may negatively affect vaccine efficacy if changes occur at major antigenic sites. Thus, continuous monitoring for novel genetic and antigenic virus types is needed. Additional studies are in progress to characterize nucleotide sequences of all CPV isolates from this case.