Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma

Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level was falsely elevated due to interference from HAs. The interfering antibodies were polyspecific immunoglobulins, with fairly broad species-specificity, which affected the eGH immunoassay by bridging the mouse IgG capture antibody and the rabbit IgG conjugate. This produced artificial sandwiches which led to overestimation of the eGH plasma concentration. Spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma of several species significantly reduced but did not totally eliminate the HAs interference. Immunoglobulins and whole plasma differed in their ability to block the interference, suggesting that HAs may recognize other proteins beside immunoglobulins in animal sera. To investigate whether HAs have any implications in equine clinical practice, we decided to seek information on the incidence of HAs interference in normal animals. We collected single plasma samples from another 114 horses and we found that 5 of these had plasma HAs. Therefore, in total 6 out of the 127 horses examined (4.7%) had plasma HAs generating falsely elevated eGH measures. In conclusion, this study provides the first evidence of HAs in horse plasma interfering with an immunoassay and indicates that veterinary surgeons and diagnostic laboratory staff should be aware of this potential for interference in tests on horse plasma using monoclonal or polyclonal antibody reagents.     

The Effect of Species-Specific FSH Administration During In vitro Maturation of Bovine Oocytes on Embryonic Developmental Capability

Veterinary Research Communications -     

Effects of growth hormone on oocyte in vitro maturation and its localization in the canine cumulus-oocyte complexes

Veterinary Research Communications -     

Cardiorespiratory effects of isoflurane anesthesia in crested caracaras (Caracara plancus)

To evaluate the cardiorespiratory changes induced by isoflurane (ISO) anesthesia in the crested caracara (Caracara plancus), eight crested caracaras that weighed 1.0 kg (range 0.9-1.1 kg) were the subjects for the study. The birds were anesthetized by face mask with ISO for brachial artery catheterization. After recovery, anesthesia was re-induced and maintained with ISO with spontaneous ventilation. Electrocardiography, direct systolic arterial blood pressure (SAP), diastolic arterial blood pressure (DAP), mean arterial blood pressure (MAP), respiratory rate (RR), end-tidal carbon dioxide (P(ET)CO2), and cloacal temperature (T degrees C) were measured before induction (baseline, under physical restraint) and after 5, 10, 15, 20, 25, 30, 35, and 40 min of ISO anesthesia. Arterial blood samples were collected for blood gas analysis at baseline, 10, 25, and 40 min. No cardiac arrhythmias were observed in the present study. RR, SAP, DAP, MAP, T degrees C and pH decreased from baseline values, whereas arterial partial pressures of oxygen and carbon dioxide, bicarbonate concentration, and P(ET)CO2 were significantly higher than baseline. Apnea was not observed in any bird. ISO anesthesia is suitable for use in healthy members of this species despite the moderate cardiovascular and respiratory depression produced.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in captive maned wolves (Chrysocyon brachyurus) from southeastern and midwestern regions of Brazil

The main purpose of the present study was to investigate the occurrence of antibodies against T. gondii and N. caninum in captive maned wolves from Brazil, considering that little information is available at the literature about infections by these parasites in this wild animal. Serum samples were obtained from 59 maned wolves originated from six zoos and from one ecological reserve of the southeastern and midwestern regions of Brazil. To detect IgG antibodies against T. gondii, an ELISA protocol was used and the results were expressed as ELISA reactivity indexes (EI). Serology for N. caninum was carried out by indirect fluorescent antibody test (IFAT) and cut-off titers were established at 1:25 dilution. From the total of the analyzed samples, 44 (74.6%) were seropositive for T. gondii and only 5 (8.5%) for N. caninum. Seropositivity for T. gondii ranged from 0 to 100% in the seven different origin locals, with rates over 50% among the six zoos, whereas no positivity was found in the samples from ecological reserve. For N. caninum, seroprevalence varied from 0 to 50% in the different locals, with the highest rates also detected in zoos. Seroprevalence for T. gondii was strongly related with age, with rates significantly higher among adult wolves (91.7%) when compared to newborn or young animals. Seropositive samples for N. caninum were found predominantly in adult wolves. For both parasites, seroprevalence did not show a significant distinction in relation to gender. Although seroprevalence for T. gondii was significantly higher when compared to N. caninum in the Brazilian captive maned wolves tested, these findings reflect the great exposure of this species to T. gondii and, in lower extension, to N. caninum. Also, the present study demonstrated for the first time the presence of antibodies to N. caninum in wild life from South America.     

Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil

Toxoplasma gondii isolates are highly diverse in domestic animals from Brazil. However, little is known about the genetics of this parasite from wild mammals in the same region. Reveal genetic similarity or difference of T. gondii among different animal populations is necessary for us to understand transmission of this parasite. Here we reported isolation and genetic characterisation of three T. gondii isolates from wild animals in Brazil. The parasite was isolated by bioassay in mice from tissues of a young male red handed howler monkey (Alouatta belzebul), an adult male jaguarundi (Puma yagouaroundi), and an adult female black-eared opossum (Didelphis aurita). The monkey and the jaguarundi had inhabited the Zoo of Parque Estadual Dois Irm?os, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in S?o Paulo State, Southeastern Brazil, and euthanised for this study because it was seropositive for T. gondii (titre 1:100 by the modified agglutination test, MAT). Ten PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, were used to genotype the isolates. T. gondii was isolated from the brain and heart homogenate of the monkey, the muscle homogenate of the jaguarundi, and the heart homogenate of the black-eared opossum. This was the first isolation of T. gondii from a neotropical felid from Brazil. The isolate from the monkey (TgRhHmBr1) was not virulent in mice, whereas the isolates from the jaguarundi (TgJagBr1) and the black-eared opossum (TgOpBr1) were virulent in mice. The genotype of the isolate from the monkey has been identified in isolates from a goat and ten chickens in the same region of Brazil, suggesting that it may be a common lineage circulating in this region. The genotypes of the isolates from the jaguarundi and the black-eared opossum have not been previously reported. Although there are already 88 genotypes identified from a variety of animal hosts in Brazil, new genotypes are continuously being identified from different animal species, indicating an extremely high diversity of T. gondii in the population.     

Molecular characterization of Cryptosporidium spp. in native breeds of cattle in Kaduna State, Nigeria

Despite numerous molecular epidemiologic studies of cryptosporidiosis in dairy cattle in industrialized countries, there are very few studies on the diversity and public health significance of Cryptosporidium species in native cattle in developing countries. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was used to detect and identify Cryptosporidium spp. in 194 fecal specimens from 2 to 365 days old calves in 20 White Fulani and Sokoto Gudali herds in Nigeria. Thirty one (16.0%) of the specimens were positive for Cryptosporidium. Restriction digestion of the PCR products showed the presence of Cryptosporidium bovis (7.2%), Cryptosporidium ryanae (4.1%), Cryptosporidium andersoni (2.5%), and concurrent occurrence of C. bovis and C. ryanae (1.5%), and C. bovis and C. andersoni (0.5%). There were no significant differences (p>0.05) in Cryptosporidium infection rates by sex, herd location, management system, breed of calves, or fecal consistency. However, calves 180 days or younger had a higher infection rate of Cryptosporidium than older calves (p=0.034). Likewise, younger calves also had higher occurrence of C. bovis and C. ryanae (p=0.022). The absence of zoonotic Cryptosporidium parvum in the calves studied suggests that native breeds of cattle may not be important in the transmission of human cryptosporidiosis in Kaduna State, Nigeria.     

Viability of Oryza sativa L. seeds stored under genebank conditions for up to 30 years

Germination ability, equilibrium relative humidity (eRH), and moisture content of ‘control’ seed samples representing 183 rice accessions stored in the active (2–4 °C) and base (?10 °C until 1993, then ?20 °C) collections of the T. T. Chang Genetic Resources Center were determined after storage for 20.5–30.5 years. Germination of seeds that had been stored in the base collection was generally high (>70 %), whereas germination was more variable for seeds stored in the active collection. Samples with lower viability after storage in the active collection were likely to have lower viability after storage in the base collection. There were significant differences in the moisture content-eRH relationship of the seeds depending on whether the seeds had been stored in the active or base collection. Based on re-test data for regular seed samples regenerated in 1979–1980 and stored in the active collection for up to 31 years, estimates of the time for ability to germinate to fall to 50 % (p ₅₀) ranged from 54 to 997 years. For the same seed samples stored in the base collection for approximately 31 years, ability to germinate has been maintained and germination increased due to improved procedures. The ability to germinate of base collection samples was also generally higher than that of ‘safety duplicate’ samples of the same seed lots that had been sent to the National Center for Genetic Resources Preservation, USA in 1981 and stored at ?18 °C. This may have been due to uptake of moisture either during processing for dispatch or as a consequence of poor packaging material. The results are discussed in relation to long-term seed storage and genebank management.