Non-immunized Calves Research Articles

Hidden antigens from third instar Hypoderma lineatum: Impact of immunization on larval survival in artificial infestations

Soluble fractions of Hypoderma lineatum third instar fat body, haemocytes and haemolymph were formulated with Quil A and used to immunize four groups of calves while a fifth group remained untreated. Calves received two subcutaneous injections of the soluble fractions, or adjuvant only delivered two weeks apart. Two weeks after the last injection the calves were exposed to 50 newly hatched larvae of H. lineatum which were placed on the skin and allowed to penetrate. Survival of larval stages was monitored by weekly palpation and collection of emergent third instars. Antibody responses to the immunogens were evaluated by immunoblots and following infestation antibody responses to first instar antigens were evaluated by an ELISA. Non-immunized calves and calves injected with adjuvant were all palpation positive for cattle grubs. In groups immunized with fat body, haemocyte and haemolymph components 100%, 33% and 33% were palpation positive for grubs respectively. First instar mortality, as reflected in palpable grubs, was high in the groups receiving injections with tissue components (99.3%, 95.1%, 95.8%, 83.9 and 80.4% mortality for those groups receiving fat body, haemocyte, haemolymph, adjuvant or control respectively). Second and third instar mortality was also higher in the immunized groups (100.0%, 91.7%, 91.7% for fat body, haemocyte, and haemolymph respectively) in comparison to the adjuvant only (14.0%) and unvaccinated (33.3%) groups. No viable flies emerged from pupae originating from larvae emergent from any of the immunized groups. Calves receiving the tissue extracts developed antibodies to several protein components following the second immunization which were still present 13 weeks post-infestation. Several proteins appeared to be common among the three tissue extracts and were recognized by antibodies from the immunized calves. All groups of calves became positive for antibodies to first instar antigens, although in some immunized calves the antibodies were transient.

Reduction of Theileria annulata Infection in Ticks Fed on Calves Immunized with Purified Larval Antigens of Hyalomma anatolicum anatolicum

Larval antigen of Hyalonmma anatolicum anatolicum, the vector of Theileria annulata, was purified by two-step affinity chromatography using anti-tick gut-specific rabbit IgG and IgG from immunized cattle. The purified antigen showed the presence of a single polypeptide of 37 kDa (GHLAgP) on SDS-PAGE. Two groups (I and II) of naive crossbred calves (Bos taurus x B. indicus) were immunized with I mg of GHLAgP in three divided doses. Immunized calves of group I were also infected with a sublethal dose of T annulata along with a group of non-immunized calves (group III). Animals in groups I, II, III as well a control group (group IV) were challenged with live nymphs of H. a. anatolicum on the 10th day of immunization. There was a significant reduction in the number of emerging adults of 56.9% +/- 1.67%, in calves of group I (p < 0.01) and 63.09% +/- 1.26% in calves of group II (p < 0.001) compared to the controls. The calves of groups I and II showed antibody responses to tick antigen up to day 70 post immunization. Infection with T. annulata was determined in the salivary glands of adult ticks that developed from the nymphs used for challenge infection. In ticks taken from group I calves, there was a 75.0% +/- 0.00% infection compared with only 85.0 +/- 2.88% infection in ticks taken from calves of group III. Using PCR, a lower infection (83.33% +/- 3.33%) was detected in ticks that developed from calves of group I compared with calves from group III (90.00% +/- 2.88%). The ground-up tick supernatants (GUTS) of the ticks taken from calves of group III yielded higher infection rate and exhibited higher infectivity titre in in vitro infection assay of bovine mononuclear cells than the GUTS of the ticks taken from calves of group I. The results suggest a partial reduction in growth rate of T. annulata in ticks feeding on calves immunized with GHLAgP.

Specific passive immunity reduces the excretion of glycoprotein E-negative bovine herpesvirus type 1 vaccine strain in calves

We investigated the excretion of either a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BHV1) vaccine strain or a conventional modified-live vaccine strain in both naı̈ve and passively immunised calves. The replication of gE-negative strain was considerably reduced in the maternally immunised calves, in comparison with the non-immune calves. On the other hand, the excretion of the gE-positive conventional vaccine strain was not reduced and even seemed to be prolonged in the presence of maternal antibodies. These results suggest that BHV1 gE may play a role in virus survival in the presence of antibodies.

Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves

Summary A noncytopathic bovine viral diarrhea virus (bvdv), bvdv-890, isolated from a yearling heifer that died with extensive internal hemorrhages, was compared for virulence in calves with noncytopathic bvdv-TGAN, isolated from an apparently healthy persistently infected calf. After challenge exposure with bvdv-890, nonimmune calves (n = 7) developed fever &gt; 40 C, diarrhea, leukopenia, lymphopenia, neutropenia, and thrombocytopenia. Most calves (n = 6) died or were euthanatized by 19 days after challenge exposure. Challenge exposure with bvdv-890 did not induce disease in 2 calves that had congenital persistent infection with bvdv or in 3 calves that had neutralizing antibody titer &gt; 4 against bvdv-890. After challenge exposure with bvdv-TGAN, nonimmune calves (n = 7) developed fever &gt; 40 C and, rarely, diarrhea or lymphopenia. All of those calves survived challenge exposure. The average maximal titer of bvdv-890 isolated from serum was 1,000 times that of bvdv-TGAN. In calves infected with bvdv-890, the average maximal percentages of lymphocytes and platelets associated with virus were greater than those found in calves infected with bvdv-TGAN. Additional findings of epidemiologic significance were prolonged shedding of virus and delayed production of viral-neutralizing antibody in 1 calf challenge-exposed with bvdv-890. Also, after production of neutralizing antibody, mutant virus that was refractory to neutralization was isolated from calves challenge-exposed with bvdv-TGAN.

A study of the basis of virulence variation of bovine rotaviruses

Rotaviruses are enteric pathogens of cattle but sub-clinical infections are common. Virulence variation has been identified with bovine rotaviruses and some rotaviruses replicated without clinical signs in non-immune calves. The rotavirus genome is composed of eleven segments of double-stranded RNA and the fourth largest segment codes for a non-glycosylated surface protein, VP4, which has been linked with virulence. In the present study the biological basis of rotavirus virulence variation was studied in vivo and compared with the known properties of the fourth gene. Calves were inoculated orally with a virulent rotavirus or a rotavirus of low virulence which multiplied but failed to cause diarrhoea. They were taken for necropsy at intervals of 2 days after inoculation. Clinical signs, virus in faeces and the percentage of infected small intestinal epithelium were determined. Damage to the small intestine was assessed by measurement of villus heights and cryptcell production rates. Virulence was associated with a greater level of colonization of the small intestinal epithelium, greater enterocyte damage and preferential infection of the upper small intestine. The fourth gene determines the ability of rotaviruses to spread in vitro and the finding that virulence was associated with greater colonization in vivo raises the possibility that this gene may have an important role in rotavirus virulence.

Induction of protective immunity by using Anaplasma marginale initial body membranes

Anaplasma marginale initial bodies of the Norton Zimbabwe strain were disrupted and separated into two membrane fractions banding at 1.15 and 1.22 g/cm3 by sucrose density centrifugation. The membrane fractions differed in their morphology and polypeptide composition. Membranes banding at 1.22 g/cm3 shared epitopes with surface-exposed polypeptides of the Florida strain of A. marginale, confirming the outer membrane location of these polypeptides. Immunization of cattle with either membrane fraction induced protection against homologous challenge, as demonstrated by significantly less anemia and lower peak rickettsemia values compared with those of adjuvant-immunized and nonimmunized calves. Protection correlated with antibody titer to membrane polypeptides. Although both membrane fractions induced protection, a 31-kDa polypeptide was the only common antigen to both fractions, as shown by reactivity of immune sera. Identification of membrane antigens capable of inducing protective immunity should facilitate development of vaccines against anaplasmosis suitable for use in Zimbabwe.

A Controlled Trial of Bovine Dialyzable Leukocyte Extract for Cryptosporidiosis in Patients with AIDS

Cryptosporidial infection causes severe diarrheal disease in patients with AIDS. Fourteen patients with AIDS and symptomatic cryptosporidiosis were treated with a specific bovine dialyzable leukocyte extract (immune DLE) prepared from lymph node lymphocytes of calves immunized with cryptosporidia or a nonspecific (nonimmune) DLE prepared from nonimmunized calves. Six of 7 patients given immune DLE gained weight and had a decrease in bowel movement frequency, with eradication of oocysts from stool in 5 patients. Six of 7 patients given nonimmune DLE showed no decrease in bowel movement and 4, no clearing of oocytes from stool; 5 continued to lose weight. Subsequently, 5 of these 7 were treated with immune DLE; 4 had a decrease in bowel movement frequency and significant weight gain, with eradication of oocytes from stool in 2 patients. Immune DLE produces sustained symptomatic improvement in patients with AIDS and active cryptosporidiosis, but lack of an appropriate cryptosporidial antigen allows only postulation that an augmentation of cellular immunity to Cryptosporidium parvum induced by immune DLE resulted in the microbiologic and clinical improvement observed.

2',5' oligoadenylate synthetase activity in bovine peripheral blood mononuclear cells following bovine herpesvirus type-1-induced respiratory disease: a prognostic indicator?

Following aerosol-challenge of nonimmune calves with bovine herpesvirus type-1 (BHV-1) the levels of 2',5' oligoadenylate (2-5A) synthetase in peripheral blood mononuclear cells (PBML) increased significantly to peak 4-5 days after BHV-1 infection. This corresponded temporally to the period of highest susceptibility to secondary bacterial infection. Ten days post virus infection, the enzyme levels had returned to baseline values. Aerosol challenge with bacteria (Pasteurella haemolytica) did not induce 2-5A synthetase activity in PBML, neither did it influence the kinetics of 2-5A synthetase induction by virus infection during a dual viral-bacterial infection. Pretreatment of animals with bovine recombinant interferons (IFNs) 0-96 h prior to virus challenge curtailed the viral infection and thus reduced the levels of 2-5A synthetase induced by endogenously produced IFN. A relationship between 2-5A synthetase levels on day 5 post virus infection and clinical outcome of the dual infection was noted. Moreover, the high 2-5A synthetase levels could be correlated with low plasma Zn levels, another indicator of clinical respiratory disease. These results indicate that these two parameters, 2-5A synthetase and plasma Zn, in combination have potential prognostic value in viral-bacterial infections of the respiratory tract.

Protection from parasite-induced weight loss by the vaccination of calves with excretory-secretory products of larval Oesophagostomum radiatum

Excretory-secretory products (ESP) were collected from in vitro maintained Oesophagostomum radiatum larvae during the period in which the larvae molt from the third to fourth larval stage. The ESP were used to immunize 13 uninfected calves which were subsequently challenged with 1.7×10 4 infective O. radiatum larvae. Worm recoveries from immunized calves were reduced 23% compared to 12 unimmunized controls, while the number of intestinal nodules was 72% greater compared to unimmunized controls; however, neither difference was statistically significant. Immunized calves had enhanced serum IgG and IgA anti-ESP antibody responses upon challenge. No differences in serum IgG 2 or IgM antibody or cellular immune responsiveness, as determined by in vitro antigen-induced lymphocyte proliferation, were seen. Vaccination with ESP did significantly protect calves from the weight loss seen in non-immunized calves. Unimmunized calves ceased gaining weight ∼5 weeks after challenge and by 10 weeks after challenge had lost an average of 4 lbs. per calf. Over the same time interval (i.e., 5–10 weeks after challenge), the immunized calves gained an average of 23 lbs. per calf. These results strongly suggest that vaccination with ESP conferred an advantage to calves that is not correlated solely with the number of worms developing from challenge infection.

Treatment of cryptosporidiosis with oral bovine transfer factor

Cryptosporidia are intestinal protozoans long known to cause diarrhea in humans, especially those with acquired immune deficiency syndrome (AIDS). When transfer factor prepared from calves which possessed delayed-type hypersensitivity to Eimeria bovis was given to nonimmune calves and mice it conferred protection against clinical infection (coccidiosis). Recent studies with oral bovine transfer factor have shown that it can confer cell-mediated immunity to humans. Based on these findings we decided to treat eight AIDS patients suffering from Cryptosporidium-associated diarrhea with transfer factor prepared from calves immune to Cryptosporidium- Prior to treatment with transfer factor, three patients had been treated with spiramycin, one patient with α-difluoromethylornithine (DFMO), and one patient with furazolidone for greater than 1 month without clinical or laboratory improvement. Following administration of transfer factor, five or eight patients exhibited a decrease in the number of bowel movements and the development of formed stools. Cryptosporidium was eradicated from the stools of four patients but two of these patients subsequently relapsed and one patient continued to have diarrhea despite the absence of Cryptosporidium in the stool. One patient has been free of diarrhea and Cryptosporidium for 2 years after discontinuation of transfer factor therapy.

Protective immunity against clinical sarcocystosis in cattle

Five experiments involving 50 calves ( Bos taurus) were conducted to determine if oral infection with sporocysts of the pathogen Sarcocystis cruzi or the non-pathogen Sarcocystis hirsuta would stimulate development of a protective immunity against illness or death from challenge infection with large numbers of S. cruzi sporocysts. Four experiments involved Holstein calves housed in individual pens in isolation buildings; one experiment involved Hereford calves on an open range. Calves infected with 50 000 to 100 000 S. cruzi sporocysts were protected from illness and death that would have resulted from challenge infections with 250 000 or 500 000 S. cruzi sporocysts given 70–252 days later. Calves treated with the anticoccidial drug amprolium from 21 to 35 days after being given an immunizing infection with S. cruzi to reduce clinical signs of the immunizing infection were also protected. Non-immunized calves and those given S. hirsuta sporocysts were not protected.

Use of apparatus for controlled infection of buffalo calves with an aerosol of Pasteurella multocida

an apparatus suitable for infection of both buffalo calves and adults with aerosolised material is described. An enclosed system was used with a continuous aerosol generation of about 48 ft. 3/minute with output fans adjusted to create a slight negative internal pressure. Animals wore a mask fitted over the nose and apparently breathed normally. The number of viable P. multocida reaching the mask, as determined by sampling with Porton liquid impingers, was affected by the relative humidity of the atmosphere. Two calves infected with a low dose developed marked symptoms; one died within 69 hours but the other recovered. Increasing the dose 10 fold or more caused death of 4 non-immune calves within 27 to 33 hours. Post-mortem findings were similar to those of natural cases. Three calves vaccinated with saline suspensions of bacteria of different antigenic composition and challenged on the same day survived for 3 to 5 days compared to 29 hours for the control.

Response of Immunized and Nonimmunized Calves to Cecal Inoculation of First-Generation Merozoites of Eimeria bovis

Four experiments were conducted, in each of which 0.4 to 1.9 billion first-generation merozoites of Eimeria bovis were inoculated by laparotomy into the ligated cecum of a nonimmunized calf and a calf immunized with 25,000 or 50,000 oocysts. These merozoites were obtained from calves killed 15 days after inoculation with 2.0 million oocysts. An initial biopsy was performed at the time merozoites were inoculated, and three or four additional biopsies were done in each calf at intervals of 3 to 24 hr during the following 2 or 3 days. The ligature was removed 1 to 2 days after inoculation, and fecal samples collected daily were examined for oocysts. Approximately 1 week after the last biopsy in each experiment, the calves were challenged with an oral inoculation of 1.0 million oocysts and were killed 19 days later. Moderate infection occurred after the inoculation of merozoites in the nonimmunized calves of Experiments 1 and 2, but only slight infections occurred in the nonimmunized calves of Experiments 3 and 4. No infection could be detected in any of the immunized calves, except for a few parasites found in one calf at 24 hr. These findings suggest that the penetration of host cells by first-generation merozoites is inhibited in immunized calves. All of the immunized calves and two of the four nonimmunized calves showed little or no infection after challenge, but the nonimmunized calves of Experiments 3 and 4 became moderately infected. These results indicate that infection caused by inoculation into the cecum of first-generation merozoites stimulates the development of immunity. Although immunity against coccidiosis is well known in both mammals and birds, little has been learned about the way in which the parasites are affected by the immune reaction. The occurrence of unusually large first-generation schizonts in E. bovis, with the resulting ready availability of merozoites, makes this species suitable for investigation of this problem. The present study was undertaken to determine whether first-generation merozoites of E. bovis inoculated into the cecum of immunized and nonimmunized calves would be able to invade the epithelium and initiate an infection, and to determine whether infections so produced would result in immunity. Received for publication 22 January 1964. * Supported in part by research grant E-2374 from the Institute of Allergy and Infectious Diseases and by a fellowship (GPM-18,586) from the Division of General Medical Sciences, Public Health Service. Published as Journal Paper No. 385, Utah Agricultural Experiment Station. t Present address: College of Veterinary Medicine, University of Illinois, Urbana, Illinois. MATERIALS AND METHODS Twelve Holstein-Friesian male calves were used in a series of four biopsy experiments. Four of these calves were used as sources of merozoites for inoculation. The methods of housing, feeding, inoculating, sampling for oocysts, collecting and concentrating merozoites, and performing the biopsies were as described previously (Hammond, Andersen, and Miner, 1963a, b). In each experiment, one calf was immunized by inoculating it when 1 to 2 weeks old with 25,000 or 50,000 E. bovis oocysts. Approximately 1 month later, this animal and a nonimmunized calf of similar age were each given a cecal inoculation with 0.4 to 1.9 billion first-generation merozoites of E. bovis. These merozoites were obtained by scraping the intestinal mucosa of a calf killed 15 days after inoculation with 2.0 million oocysts. More than 90% of these oocysts were E. bovis; the remainder consisted chiefly of E. ellipsoidalis and E. auburnensis. Gametocytes of these two species were seen, in addition to E. bovis schizonts, in the intestinal sections of the calves used as sources of merozoites. However, the cecal inoculations resulted in pure E. bovis infections, as shown by the finding of oocysts of this species only in the fecal samples. In each experiment equal numbers of merozoites were introduced by laparotomy into the ceca of the two calves. Three or four additional biopsies

The life cycle stages of Eimeria bovis affected by the immune reaction in calves

Three experiments were conducted to determine whether schizonts of E. bovis are appreciably influenced by the immune reaction in calves. Each experiment involved two groups each of four to six calves. Each calf in one group was given an immunizing inoculation of 60,000 to 100,000 oocysts, and after 30 to 35 days each calf in both groups was given a challenging inoculation of 800,000 to 5 million oocysts. One calf in each group was killed on successive days beginning 15 to 17 days after the second inoculation. Numbers of schizonts in the small intestine were estimated by examination with a dissecting microscope and frequency of sexual stages in the cecum and colon was observed in sections. No consistent difference was found in concentration, estimated total number, or size of schizonts in the two groups, although non-immunized calves had a much higher frequency of sexual stages. The results indicate that the schizonts and preceding stages are not appreciably influenced by the immune reaction, so that immunity must chiefly affect the merozoites or sexual stages.