Lameness in Early Lactation Is Associated with Lower Productive and Reproductive Performance in a Herd of Supplemented Grazing Dairy Cows.

The main aim of this study was to assess the associations between the timing of lameness clinical case occurrence in lactation with productive and reproductive performances in grazing Holstein cows. A cohort study was carried out on a dataset with records from a commercial dairy herd (Buenos Aires, Argentina) for cows that calved and were dried off from January 2010 through June 2017. The first recorded event of lameness per lactation was considered for the study. Criteria for lactation inclusion included not having uterine diseases, mastitis, or anovulatory cysts during the studied risk period (i.e., up to 200 DIM). Therefore, a total of 7156 out of 20,086 lactations were included in the statistical analysis. The association between lameness case occurrence in lactation (cows not lame (LG0) vs. lame cows between parturition and first service (LG1) vs. lame cows between first service and first pregnancy (LG2)) with productive (i.e., accumulated milk yield to 150 DIM (MILK150) and 300 DIM (MILK305)) and reproductive performances (hazard of insemination and pregnancy) was analyzed with linear regression models and proportional hazard regression models, respectively. Lame cows produced 161 and 183 kg less MILK150 and MILK305 than non-lame herd mates, respectively. Moreover, LG1 cows produced 216 kg less MILK150 and 200 kg less MILK305 than LG0 cows, and LG2 cows also produced 58 kg less MILK150 and 158 kg less MILK305 than LG0 cows. The LG1 cows had a lower hazard of service than LG0 cows (HR = 0.43, 95%CI = 0.39-0.47). Furthermore, LG1 cows had a lower hazard of pregnancy than LG0 cows (HR = 0.52, 95%CI = 0.46-0.59) and took longer to get pregnant than LG0 cows (median [95%CI], 139 [132-144] vs. 101 [99-103]). Moreover, LG2 cows had a much lower hazard of pregnancy than LG0 cows (HR = 0.08, 95%CI = 0.05-0.12) and much longer calving to first pregnancy interval than LG0 cows (188 [183-196] vs. 101 [99-103]). In conclusion, cows that become lame in early lactation produce less milk and have lower hazards of insemination and pregnancy than herd mates that are healthy or become lame later in lactation. In addition, cows that become lame immediately after the voluntarily waiting period have the poorest reproductive performance (i.e., they have the lowest hazard of pregnancy and the longest calving to pregnancy interval).

Analysis of puerperal metritis treatment records in a grazing dairy farm in Argentina.

OBJECTIVE: To assess the efficacy of antibiotic usage for the treatment of puerperal metritis (PM) and its association with reproductive performance, a retrospective cohort study including a total of 9168 records of cows from a dairy farm in Argentina was run. MATERIAL AND METHODS: Cows having a PM3 (metricheck, scale 0-3) and treated with ceftiofur (ceftiofur crystalline free acid, 6.6 mg/kg) at 0-21 days postpartum (p. p.) (n = 2688), and cows having a PM 1-2 and not treated with an antibiotic at 0-21 days p. p. (n = 6480) were included in the study. All cows were reexamined with metricheck to assess the clinical cure (vaginal discharge [VD] score 0), partial cure (VD score similar or lower than previous), no cure (VD score higher than previous). Cows with a metricheck VD1-3 after 0-21 days p. p. were diagnosed as clinical endometritis (CE) 1-3. The occurrence of PM1-3, cure rate, calving to conception interval, the hazard of pregnancy, odds for non-pregnancy, and odds for CE were analyzed using SAS software. RESULTS: A total of 8876 PM1-3 records were included, 2435 records of PM3 treatments with ceftiofur (27.43 %), and 6441 records of PM1-2 (72.57 %) with no treatment. Cows having PM1 and PM2 became pregnant 14 and 12 days earlier than cows with PM3 (p < 0.001). The PM3 ceftiofur treated cows had a clinical cure of 24.85 % (PM0); 53.63 % had a partially cure; and 18.52 % no cure. Conversely, cows with PM1-2 had a 51.96 %, 20.70 %, and 24.53 % cure rate, respectively (p < 0.001). Cows having complete cure became pregnant 13 and 11 days earlier than cows having partial cure and no cure (p < 0.001). Cows that had PM3 during the first 21 days p. p. had twice the chances of developing CE compared to cows having PM1-2 (41.28 % vs. 24.14 %, p < 0.001). After 21 days p. p., less than 1 % of cows with clinical cure developed CE compared to 63.32 % that developed CE with partial cure, and 38.21 % with no cure (p < 0.001). CONCLUSION AND CLINICAL RELEVANCE: After ceftiofur treatment, 78 % of cows were cured when measured by disappearance of fetid VD but only 25 % of cows had clinical cure when measured by appearance of a clear VD. The cows that remained with clinical metritis had more chances of having CE after 21 days p. p. and had more days open than cows with clear normal VD.

Respuesta inmunoprotectora inducida por la glicoproteina D recombinante en el modelo de infección respiratoria de ratones BALB/c por alfaherpesvirus equino 1 / Immunoprotective response induced by recombinant glycoprotein D in the BALB/c respiratory mouse model " of Equid alphaherpesvirus 1 infection

Equid alphaherpesvirus 1 (EHV-1) infection causes abortion, respiratory disease, perinatal deaths and neurological disorders in horses. The natural infection and available vaccines provide only partial and short-lived protection against reinfections. In the present study, we analyzed the ability of purified baculovirus-expressed glycoprotein D (gD) administered by different routes to induce protective immunity in BALB/c mice after challenge with the EHV-1 AR8 strain. Clinical signs varied among the different groups of mice immunized by parenteral routes, and, although gD induced a specific serum IgG response, it did not prevent the virus from reaching the lungs. Intranasally immunized mice showed no clinical signs, and virus isolation from lungs, histological lesions and antigen detection by immunohistochemistry were negative. In addition, by this route, gD did not stimulate the production of serum IgG and IgA. However, a specific IgA response in the respiratory tract was confirmed in intranasally immunized mice. Thus, we conclude that the mucosal immune response could reduce the initial viral attachment and prevent the virus from reaching the lungs. Our findings provide additional data to further study new immunization strategies in the natural host.

La infección con alfaherpesvirus equino 1 (EHV-1) causa abortos, enfermedad respiratoria, muertes perinatales y desórdenes neurológicos en equinos. La infección natural y las vacunas disponibles solo proporcionan protección parcial y de corta duración contra las reinfecciones. En el presente estudio se analizó la inducción de inmunidad protectiva de la glicoproteina D (gD) expresada en baculovirus y purificada al ser administrada por diferentes rutas en ratones BALB/c desafiados con la cepa AR8 de EHV-1. Los signos clínicos fueron variables entre los grupos de ratones inmunizados por rutas parenterales y, aunque la gD indujo respuesta especifica de IgG en suero, no logró prevenir la llegada del virus al pulmón. En los ratones inmunizados intranasalmente no se observaron signos clinicos ni lesiones histopatológi-cas, y el aislamiento viral y la detección de antigenos por inmunohistoquímica en pulmón fueron negativos. Además, por esta ruta la gD no estimuló la producción de IgG y de IgA en suero. Sin embargo se confirmó la respuesta de IgA especifica en el tracto respiratorio de ratones inmunizados intranasalmente. Esta respuesta inmune mucosal podría haber reducido la unión inicial del virus a la célula huésped y, de este modo, prevenir la llegada del virus al pulmón. Nuestros hallazgos proporcionan un aporte para continuar estudiando nuevas estrategias de inmunización en el huésped natural.

Detección y caracterización molecular de Chlamydia psittaci y Chlamydia abortus en psitácidos mascotas en la provincia de Buenos Aires, Argentina / Detection and molecular characterization of Chlamydia psittaci and Chlamydia abortus in psittacine pet birds in Buenos Aires province, Argentina

In order to determine the presence and genetic diversity of Chlamydia spp. in the north-eastern area of Buenos Aires province, Argentina, conjunctival, oropharyngeal, cloacal swab and tissues were collected from a total of 90 psittacine pet birds of different age and clinical manifestations. Through molecular methods, Chlamydiaceae was detected in 30% (27/90) of the samples, out of which 70.3% (19/27) were positive for Chlamydia psittaci and 14.9% (4/27) for Chlamydia abortus. Nine C. psittaci positive samples were genotyped by ompA gene sequences, 8 clustered within genotype A and 1 within genotype B. A significant association was observed between the presence of Chlamydia spp. and the manifestation of clinical signs compatible with chlamydiosis, as well as with the age of the birds (younger than one year old). This report contributes to the improvement of our understanding of chlamydial agents in our country.

Con el objetivo de determinar la presencia de Chlamydia spp. en psitácidos del área noreste de la provincia de Buenos Aires y conocer su diversidad genética, se recolectaron y analizaron mediante métodos moleculares hisopados conjuntivales, orofaríngeos, cloacales y tejidos de un total de 90 psitácidos de diferentes edades y con diversas manifestaciones clínicas. El 30% (27/90) de las muestras procesadas fueron positivas para Chlamydiaceae; el 70,3% (19/27) de estas resultaron positivas para Chlamydia psittaci y el 14,9% (4/27) para Chlamydia abortus. Nueve muestras positivas para C. psittaci fueron genotipificadas por secuenciación del gen ompA: 8 correspondieron al genotipo Ay una al genotipo B. Se observó una asociación significativa entre la presencia de Chlamydia spp. y la manifestación de signos clínicos compatibles con clamidiosis, como así también con la edad de las aves (menores de un ano). Este informe contribuye a mejorar nuestro conocimiento de los agentes clamidiales en nuestro país.

Detection and molecular characterization of Chlamydia psittaci and Chlamydia abortus in psittacine pet birds in Buenos Aires province, Argentina.

In order to determine the presence and genetic diversity of Chlamydia spp. in the north-eastern area of Buenos Aires province, Argentina, conjunctival, oropharyngeal, cloacal swab and tissues were collected from a total of 90 psittacine pet birds of different age and clinical manifestations. Through molecular methods, Chlamydiaceae was detected in 30% (27/90) of the samples, out of which 70.3% (19/27) were positive for Chlamydia psittaci and 14.9% (4/27) for Chlamydia abortus. Nine C. psittaci positive samples were genotyped by ompA gene sequences, 8 clustered within genotype A and 1 within genotype B. A significant association was observed between the presence of Chlamydia spp. and the manifestation of clinical signs compatible with chlamydiosis, as well as with the age of the birds (younger than one year old). This report contributes to the improvement of our understanding of chlamydial agents in our country.

Immunoprotective response induced by recombinant glycoprotein D in the BALB/c respiratory mouse model of Equid alphaherpesvirus 1 infection.

Equid alphaherpesvirus 1 (EHV-1) infection causes abortion, respiratory disease, perinatal deaths and neurological disorders in horses. The natural infection and available vaccines provide only partial and short-lived protection against reinfections. In the present study, we analyzed the ability of purified baculovirus-expressed glycoprotein D (gD) administered by different routes to induce protective immunity in BALB/c mice after challenge with the EHV-1 AR8 strain. Clinical signs varied among the different groups of mice immunized by parenteral routes, and, although gD induced a specific serum IgG response, it did not prevent the virus from reaching the lungs. Intranasally immunized mice showed no clinical signs, and virus isolation from lungs, histological lesions and antigen detection by immunohistochemistry were negative. In addition, by this route, gD did not stimulate the production of serum IgG and IgA. However, a specific IgA response in the respiratory tract was confirmed in intranasally immunized mice. Thus, we conclude that the mucosal immune response could reduce the initial viral attachment and prevent the virus from reaching the lungs. Our findings provide additional data to further study new immunization strategies in the natural host.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies.

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.

Development of a peptide ELISA for the diagnosis of Equine arteritis virus.

A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.

Production of equine herpesvirus 1 recombinant glycoprotein D and development of an agar gel immunodiffusion test for serological diagnosis.

Equine herpesvirus 1 and 4 (EHV-1 and 4) infect most of the world's horses, causing serious clinical illness. Viral glycoproteins have been identified as the immunodominant antigens that generate the antiviral serological responses to EHV-1 and EHV-4 in infected horses. Here, glycoprotein D of EHV-1 was expressed by a recombinant baculovirus, purified and evaluated by a simple agar gel immunodiffusion test (AGID). Compared with virus neutralization, serological analysis by AGID showed good specificity (100%) and sensitivity (99.5%). The estimated Kappa values for repeatability and reproducibility were satisfactory. Thus, this rapid, inexpensive, simple and highly specific AGID test seems to be a valuable alternative tool for serological detection of antibodies against both EHV-1 and EHV-4.

Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs.

Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky's disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine.

Hematology and serum biochemistry of free-ranging nutria (Myocastor coypus).

Information on reference blood values in the literature is lacking for many wild rodents. In this study, comprehensive reference intervals (RIs) for a wide range of analytes from 101 healthy free-ranging nutria were determined. Animals were captured in Buenos Aires, Argentina (37degrees 50'S, 57 degrees 34'W), and southward (38 degrees 60'S, 58 degrees 23'W), encompassing major biotopes of agricultural pampas with dunes and grassland steppes on the east coast. Traps were set at locations with high-density nutria populations (i.e., those areas that showed signs of movement, territorial marking, or feeding activities). Although the small sample size limits the interpretation of these findings, RIs were determined by a robust method using the central 95th percentile. In nutria, the RI range varied greatly for the leukocyte differentials, with mature neutrophils: 3,907-5,544/mmicrol for females and 3,744-5,900/microl for males; band neutrophils: 0-10/ll for females and 3-18/microl for males; lymphocytes: 4,213 5,940/microl for both sexes combined; monocytes: 165-402/microl for both sexes combined; eosinophils: 13-91/microl for females and 108-165/microl for males; and basophils: 0-87/microl for both sexes combined. Platelet concentration was 543-727 x 10(9)/L for both sexes combined. There was also a wide RI range for biochemistry values for some enzymes, such as alkaline phosphatase: 200-399 IU/L for both sexes combined; cholinesterase: 762-1,407 IU/L for females and 763-1,284 IU/L for males; creatine kinase: 182-552 IU/L for females and 162-451 IU/L for males; amylase: 853-1,865 IU/L for females and 779-1,293 IU/L for males; and glucose concentration 120.2-180.6 mg/dl for both sexes combined. Conversely, there was not a wide pooled RI range for calcium: 7.0-11.2 mg/dl; phosphorous: 6.1-9.3 mg/dl; sodium: 133.0-159.0 mEq/L; potassium: 3.0-8.2 mEq/L; chloride: 101.4-143.0 mEq/L; and urea: 11.3-36.8 mg/dl. The red blood cell indices had a narrow range, with mean corpuscular volume: 84.0 -102.5 fl and mean corpuscular hemoglobin concentration: 18.2-28.8 g/dl, and which was most likely due to strict physiologic controls. The results from this study were similar to those previously reported for farmed nutria.

A differential ELISA based on recombinant immunodominant epitopes of the gE gene of SHV-1 in a baculovirus-insect cell system to discriminate between pigs infected naturally with pseudorabies and vaccinated pigs.

In the present study, the fragment corresponding to the immunodominant epitopes of the gE gene (gEpi) from the CL15 Argentinean strain of pseudorabies virus was expressed successfully in a baculovirus-insect cell system that contained the M6 gene of Bluetongue virus, which encodes the NS1 nonstructural protein. This protein has the ability to polymerize into highly immunogenic tubules inside infected cells that can be purified at large quantities by ultracentrifugation. Previously, the NS1 protein has been expressed by fusing it to sequences derived from viruses, such as human immunodeficiency virus type 1, hepatitis B virus, bovine leukemia virus, foot-and-mouth disease virus and influenza A virus. In the present study, a recombinant protein was obtained containing the gEpi fused to NS1 (NS1-gEpi) and used it as ELISA antigen for detection of anti-gE antibodies in order to discriminate between infected and vaccinated animals. This is the first report where gEpi was expressed in this particular baculovirus-insect cell system.

Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies.

The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mouse-horseradish peroxidase conjugate and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.