Reproductive immunology in viviparous mammals: evolutionary paradox of interactions among immune mechanisms and autologous or allogeneic gametes and semiallogeneic foetuses.

Literally, reproductive immunology was born in bovine on-farm reproduction where seminal experiments intended for developing methods for embryo transfer in cattle were performed. Actually, these experiments led to two of major concepts and fundamental principles of reproductive immunology using the bovine species as a model for biomedical research, namely the concept of acquired immunological tolerance and the paradox of the semiallogeneic bovine foetus whereby such organism can develop within an immunologically competent host. Peter Medawar, a scientist who together with Frank Macfarlande Burnet shared the 1960 Nobel Prize in physiology or medicine for discovery of acquired immunological tolerance, while studying dizygotic cattle twins, thereby giving birth to reproductive immunology. Also, these findings significantly influenced development of organ transplants and showed that using farm animals as models for studying transplantation immunology had general relevance for mammalian biology and health including those of humans. However, the interest for further research of the fascinating maternal immune influences on pregnancy and perinatal outcomes and of the prevention and treatment of immunologically mediated reproductive disorders in viviparous mammals of veterinary relevance by veterinary immunologists and reproductive clinicians have been very scarce regarding the application of nonspecific immunomodulatory agents for prevention and treatment of subfertility and infertility in pigs and cattle, but still broadening knowledge in this area and hold great potential for improving such therapy in the future. The aim of the current overview is to provide up-to-date information and explaining/translating relevant immunology phenomena into veterinary practice for specialists and scientists/clinicians in reproduction of animals.

Effects of dietary clinoptilolite on reproductive performance, serum progesterone and insulin-like growth factor-1 concentrations in dairy cows during pregnancy and lactation.

The objective of this study was to evaluate the effects of dietary zeolite clinoptilolite on re- productive performance, serum progesterone and insulin-like growth factor 1 (IGF-1) concentra- tions in 78 Holstein Friesian (HF) cows during pregnancy and lactation. The cows were divided into two groups comprising 40 (control group; CON) and 38 (CPL group) cows. To assess repro- ductive performance of HF cows the following variables were registered: the interval from calv- ing to first insemination (days open to first service, DFS), the interval from calving to pregnancy (days open to pregnancy, DOP), and the number of services per pregnancy (NSP). The average values of progesterone (5.64±0.59 ng/mL vs. 5.16±0.64 ng/mL) were not statistically different (p⟨0.05) and IGF-1 levels (400.17±17.72 ng/mL vs. 348.36±20.39 ng/mL) were higher in the CON than in the CPL group which received 50 g of clinoptilolite twice a day. However, in the CPL group ovarian cyclity resumed on days 40 and 60 postpartum. In addition, DFS (p⟨0.05) and DOP (p>0.05) were shorter in the CPL than in the CON group (115.1±19.9 and 137.5±36.3 days vs. 124.2±17.3 and 143.8±33.5 days, respectively). During 305 days of lactation, milk production was higher in the CPL vs. CON (8325.5±628.8 kg vs. 8050±586.8 kg). The NSP was lower in the CPL than in CON group (1.91 vs. 2.14). The dietary clinoptilolite supplement had a positive in- fluence on milk yield, exhibited modulating effects on endocrine status of dairy cows, and im- proved reproductive performance, with the decreased NSP, and fewer DFS and DOP.

The onset of puberty in Cameroon Dwarf goats kept as pets in northwestern Croatia.

The aim of this study was to determine onset of puberty in Cameroon Dwarf goats (CDGs) kept as pets in northwestern Croatia by determining progesterone (PGS) and insulin-like growth factor-I (IGF-I) levels in the blood by RIA methods. The first cyclic ovarian activity was estimated according to hormone profiles as determined in CDG in a moderate climate environment. Sixteen female CDG kids were kept in stables with access to pasture which provided space for exercise. The goat kids born in winter (December-January-February), spring (March-April-May), summer (June-July-August) and autumn (September-October-November) were assigned into four groups according to the season of the year of birth (n = 4 in each group). At 75 days of age at the initiation of the study, they weighed between 3.2 and 5.1 kg (4.24 ± 0.53 kg). The onset of ovulatory activity was determined by PGS and IGF-I serum concentrations every 10 days starting from 75 days to 155 days of age. The onset of puberty in CDG kids occurred on average at 141.15 ± 2.66 days of age, but varied depending on the season of birth. All CDG kids born during summer were in heat by 155 days of age. In the other groups (kids born in autumn, winter or spring), one goat in each group was not in heat. Changes in blood serum PGS and IGF-I concentrations during prepubertal and pubertal periods could aid in the evaluation of reproductive status and determination of the onset of puberty in CDG during all seasons of the year in a moderate climate region.

Two outbreaks of neuropathogenic equine herpesvirus type 1 with breed-dependent clinical signs.

Equine herpesvirus type 1 (EHV-1) is a worldwide spread pathogen of horses. It can cause abortion, respiratory and neurological disease and consequentially significant economic losses in equine industries. During 2009, two outbreaks of EHV-1 were confirmed in two stud farms in Eastern Croatia. The first outbreak occurred in February following the import of 12 horses from USA, serologically negative to EHV-1 before transport. Four mares aborted in the late stage of pregnancy and one perinatal death was recorded. Other six mares showed clinical signs of myeloencephalopathy with fatal end in four. One month later, the second EHV-1 outbreak was confirmed in stud farm about 100 km further with 17 abortions, three perinatal deaths and one mild neurological case. Epidemiological data showed that the disease was probably introduced in the first stud farm during international transport. The second outbreak started with the introduction of clinically healthy stallion from the first stud farm. Molecular characterisation and phylogenetic analysis confirmed that, despite different clinical signs, the identical virus caused both outbreaks. Both horse populations were free from EHV-1 infection before the outbreak and had not been vaccinated. Significant difference in clinical signs could be explained by different breed-related risk factors.

Serum antibodies directed against classical swine fever virus and other pestiviruses in wild boar (Sus scrofa) in the Republic of Croatia.

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country. The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.

An outbreak of Salmonella Abortusovis abortions in sheep in south Croatia.

The aim of the study was to give an account of the epidemic of abortions in sheep caused by Salmonella enterica ssp. enterica serovar Abortusovis, which occurred in Dalmatia, south Croatia, in winter 2003-2004. Five sheep flocks with rate of abortion ranging from 22% to 38% during the last-third of gestation were examined. Salmonella Abortusovis was isolated from 13 vaginal smears and two fetuses. Direct inoculation was found to be superior to pre-enrichment and enrichment in selective broth for Salmonella Abortusovis isolation. The isolates were biochemically identified, and characterized by serotyping and polymerase chain reaction based on the amplification of the IS200 sequence specific for Salmonella Abortusovis. A fragment of 900 bp was detected in all Salmonella Abortusovis isolates. The sensitivity testing of the isolates, carried out by the disk diffusion method and the determination of the minimal inhibitory concentrations, resulted in a high sensitivity to almost all antimicrobials used. Only two isolates were moderately sensitive to oxytetracycline, whereas one isolate showed resistance to streptomycin. Campylobacter fetus ssp. fetus and Listeria monocytogenes were excluded as causative agents of abortion in sheep by culture testing, and brucellosis, leptospirosis, Q fever and chlamydiosis by serological testing.

Immune complex-based vaccine for pig protection against parvovirus.

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.

Cortisol and immune measures in boars exposed to three-day administration of exogenous adrenocorticotropic hormone.

The aim of the study was to evaluate the effect of adrenal stimulation by adrenocorticotropic hormone (ACTH) on blood cortisol concentration and on circulating total and differential leukocyte counts during and in the 16 days after ACTH administration. Swedish Landrace boars aged approximately 6-7 months were used. ACTH-treated animals (n = 7) were given ACTH intravenously at 10 microg/kg body mass for 3 days. A control group of animals (n = 7) received 1 ml of sterile 0.9% saline intramuscularly. ACTH induced a highly significant increase (p>0.0001) in serum cortisol in treated boars. On the day after the last ACTH dose, the cortisol concentration was significantly higher, but the level of significance was lower than during ACTH administration (p>0.05). During ACTH treatment, a significant increase was recorded in total leukocyte count and neutrophil percentage (p>0.05 to p>0.0001), along with the increase in blood cortisol concentration, whereas percentage lymphocyte count showed a significant decrease. Lymphopenia disappeared upon cessation of treatment, but neutropenia developed in the week after treatment. On all three days of ACTH challenge, the neutrophil-to-lymphocyte ratio was significantly increased. An increase in eosinophil percentage was recorded on treatment days 1 and 2, whereas ACTH treatment had no effect on basophil percentage. In conclusion, three-day administration of ACTH to young boars during restraint caused effects similar to acute stress situations, as suggested by disappearance of the effects on immune function after the last drug dosage.

Effect of ACTH administration on biochemical and immune measures in boars.

The effects of stress induced by adrenocorticotropic hormone (ACTH) on biochemical and immune changes in Swedish Landrace boars aged approximately 6-7 months were observed during ACTH administration and for 16 days after the cessation of treatment. Adrenocorticotropic hormone treated animals (n = 7) were given intravenously 10 microg/kg body mass of ACTH for 3 days. Control group of animals (n = 7) received intramuscularly 1 ml of sterile 0.9% saline. Total protein and globulin levels were significantly elevated during the induced stress period and for the next 16 days (P < 0.01 to P < 0.0001, respectively). Also, serum immunoglobulin IgG concentration was significantly elevated during and after ACTH injection (P < 0.001 to P < 0.0001). Adrenocorticotropic hormone treatment had no effect on serum albumin, IgA and IgM concentrations. Glucose concentration was significantly decreased on the second day of ACTH administration (P < 0.001) and on day 9 after treatment (P < 0.001). Calcium level was significantly decreased only for 24 h after the last ACTH dosage (P < 0.01). Also, serum phosphorus level was significantly decreased on the first (P < 0.05) and third (P < 0.001) days of ACTH challenge but remained unaffected after the cessation of ACTH treatment. It is concluded that the administration of ACTH to boars results in immune humoral and biochemical changes during stress and the post-stress period.

Postweaning multisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2).

The objective of this study was to characterise porcine circovirus type 2 (PCV2) from pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS) in Croatia, and to determine the epizootiological, clinical and pathomorphological features of the disease. During a systematic health monitoring programme conducted in the period from January 2002 to June 2003, PMWS was suspected on eight different pig-producing farms in Croatia. The diagnosis of PMWS met all three key criteria: the presence of compatible clinical signs, the presence of the characteristic microscopic lymphoid lesions, and the detection of PCV2 within the lesions by polymerase chain reaction (PCR) and by in situ hybridisation (ISH). Moreover, PCV2 DNA from swine tissues was extracted and sequenced. The phylogenetic analysis of 4 Croatian PCV2 strains showed close relationship to PCV2 strains isolated in Slovenia, France, the Netherlands, the United Kingdom, China and Hungary. PCV2 was also demonstrated by electron microscopy in the lymph node of an affected animal. This is the first demonstration of PMWS in Croatia based on all scientifically accepted diagnostic criteria.

Leukocyte subsets and specific antibodies in pigs vaccinated with a classical swine fever subunit (E2) vaccine and the attenuated ORF virus strain D1701.

Total white blood cell (WBC) counts and percentages of CD4a+, CD8a+, CD5a+, CD45RA+, CD45RC+, wCD21+ and SWC3a+ cells in the peripheral blood of pigs were analysed in this study. Blood samples were collected before and on days 4, 10, 21 and 28 after vaccination. Group 1 pigs were vaccinated with a subunit E2 vaccine (gp E2 32 microg/dose), and Group 2 received a subunit vaccine combined with an attenuated ORF virus strain D1701 10(6.45) TCID50/dose. Control pigs received a placebo. The total WBC count and percentage of particular cell types were within the normal range in vaccinated and control pigs. Although the mechanism of attenuated ORF virus activity is not clear, changes were observed in CD4a+, CD5a+, CD8a+, CD45RA+ and CD45RC+ cells in pigs that received the combination of a subunit vaccine and ORF virus. However, the percentage of wCD21+ and SWC3a+ did not differ significantly from that recorded in pigs given only the subunit vaccine. At days 4 and 10 the number of pigs positive to E2 antibodies was higher in the group that received the subunit vaccine and ORF virus than in pigs vaccinated with the subunit vaccine only. A higher percentage of memory cells (CD45RC+) as well as Th and Tc lymphocytes in pigs that received the ORF virus and the subunit vaccine could be ascribed to a nonspecific influence of the ORF virus on the development (through cognate interactions between T and B cells) and the duration (presumed according to the finding of the clonal expression of memory cells) of humoral immunity (assessed by a higher number of seropositive pigs in this group). This seems likely since the proportion of these cells was found to be lower in the pigs that received E2 vaccine only.

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia.

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.

Serological evidence of bluetongue and a preliminary entomological study in southern Croatia.

In December 2001, bluetongue (BT) was confirmed serologically by the Croatian Veterinary Institute using the competitive enzyme-linked immunosorbent assay (c-ELISA). Results of the serological testing of blood samples from ruminants in the Dubrovacko-Neretvanska County are presented (3,318 sera of ruminants from 53 herds were examined). In total, 357 bovine sera (178 or 49.9% positive), 1,268 ovine sera (174 or 13.7% positive) and 1,693 caprine sera (270 or 15.9% positive) were tested. Antibodies to BT virus serotype 9 were detected in 212 of the positive sera by serum neutralisation. A preliminary light-trap survey for midges of the Culicoides genus was also performed in the Dubrovacko-Neretvanska County. Fourteen light-trap collections from seven locations were examined and yielded a total of 4,872 Culicoides of which 4,492 (92%) belonged to the Obsoletus Complex (including C. obsoletus and C. scoticus).

Wild boars (Sus scrofa) as reservoirs of Brucella suis biovar 2 in Croatia.

This work presents the results of findings for brucellosis in wild boars and domestic swine in two regions of Croatia. In the region of Djakovo the blood samples of 211 wild boars were analysed and in 29.4% of the samples serologically positive reactions were established. In the same region the blood samples of 1080 domestic swine on pastures were also analysed and positive serological reactions were established in 12.3%. In the regions around Lonjsko Polje the blood samples of 53 wild boars were analysed and in 22.6% of them positive serological reactions were established. On several locations around Lonjsko Polje the blood samples of 901 domestic swine were serologically analysed and 13.5% of the swine were found to be seropositive. Bacteriological analyses of submitted materials from 24 wild boars resulted in isolation of Brucella from seven (29.2%) samples, and from 43 samples originating from domestic swine that had aborted and had been serologically positive, Brucella were isolated from 25 (58.1%) swine, as well as from 10 (62.5%) out of 16 aborted piglets. In all the isolates Brucella suis biovar 2 was identified. Wild boars are carriers and reservoirs of Brucella suis biovar 2 in Croatia.

Genetic typing of recent classical swine fever virus isolates from Croatia.

During a period of 5 years (1997-2001) several outbreaks of classical swine fever (CSF) were recorded in Croatia. For genetic typing, fragments of 150 nucleotides within the 5'-non-translated region (5'-NTR) and 190 nucleotides within the E2 glycoprotein coding gene of nine field isolates that were derived from domestic pigs and wild boars were used. For better epizootiological understanding, isolates from other European countries were included in the study. The results show that the isolates belong to subgroups 2.1 and 2.3 of CSF virus. Isolates from subgroup 2.1 were collected from domestic pigs during sporadic outbreaks in June 1997 and are genetically closely related. A genomic similarity between these isolates and CSF virus isolates from pigs in other European countries from the same year could also be confirmed. In contrast, the isolate from October 1997 was found to be a member of subgroup 2.3, and is closely related to European CSF virus isolates from outbreaks in the last decade in Western and Central European countries. These results show that two different sources of CSF virus caused outbreaks in Croatia during the same year. Furthermore, a close relationship was found between an isolate from a domestic pig in 1999 and isolates of subgroup 2.3 that originated from Croatian wild boars.

Proportions and phenotypic expression of peripheral blood leucocytes in pigs vaccinated with an attenuated C strain and a subunit E2 vaccine against classical swine fever.

The influence of an attenuated classical swine fever virus C strain vaccine and a subunit E2 vaccine against classical swine fever on the peripheral blood leucocyte proportion and phenotypic expression in 12-week-old pigs was studied. The C strain was amplified in minipig kidney cell culture and final product contained 10(4 +/- 0.15) TCID50/ml, while the subunit vaccine contained 32 microg per dose of gp E2. Haematological findings showed that the vaccines did not cause leucopenia or lymphocytopenia and the number of neutrophils and eosinophils during the observation period was within physiological range. The results of the proportion of CD4a+, CD5a+, CD8a+, wCD21+, CD45RA+, CD45RC+ , non-T non-B, SWC3a+ and CD11b+ cells were gained by single-colour flow cytometry. At the end of the trial a significantly increase of percentage of CD4+, CD5a+, CD8+, wCD21+ cells has been found in pigs that received the subunit vaccine and the percentage of CD4+, CD5a+, CD8+, CD45RA+ and CD45RC+ cells was higher in pigs that received the attenuated vaccine. Twenty-eight days after vaccination the percentage of CD4+, CD45RA+ and CD45RC+ was significantly higher in pigs vaccinated with the C strain than in pigs vaccinated with the subunit vaccine. In contrary, the percentage of the wCD21- cells was higher in pigs that received the subunit vaccine. Statistically higher values of SWC3a+ and lower values of CD11b+ cells was observed in pigs that received the attenuated vaccine than in pigs vaccinated with the subunit vaccine. Taken altogether, our results showed that the subunit vaccine produced a better stimulation of B cells and CD11b+ monocytes/macrophages /granulocytes/NK cells, whereas the attenuated vaccine induced a higher response of Th cells, naive/memory cells and macrophages/neutrophils. Thus, both vaccines were able to influence the porcine immune system, by activating different subsets of the immune effector/accessory cells.

Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever.

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 microg of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10(4+/-0.15) TCID50/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre > 1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.

Immunophenotyping of leukocyte subsets in peripheral blood and palatine tonsils of prefattening pigs.

The quantitative and distribution patterns of porcine peripheral blood and tonsillar lymphoid/myeloid cell subsets were assessed in order to establish the immune status of farm pigs prior to their transfer to fattening units. Peripheral blood and tonsillar samples were taken from clinically healthy, nonvaccinated, 12-week-old pigs, either ex vivo or following euthanasia. Single-colour flow cytometry, using monoclonal antibodies (mAbs) reactive with the swine leukocyte cluster of differentiation (CD) antigens, gave the proportions of lymphoid (9.7% CD4+, 8.0% CD8+, 36.9% CD5a+, 20.3% CD16+, 6.9% CD21+, 86.3% CD45+, 41.8% CD45RA+, 48.3% CD45RC+), null cells (6.9%) and myeloid cells (23.7% CD11b+ and 5.4% SWC3a+) in peripheral blood. In situ identification and distribution of lymphoid cells in the tonsils (CD3a+, CD21+, CD45RA+, CD45RC+) was performed with anti-CD mAbs using the avidin-biotin complex method. Most CD3a+ cells were in the parafollicular areas, with many cells in the follicles. CD21+ cells were scattered throughout the parafollicular area, with only a few cells inside lymphoid follicles. CD45RA+ cells were mostly concentrated in the follicles but many positive cells were present in the parafollicular area. Many CD45RC+ cells were visible in the parafollicular area, a few positive cells were in the crypt epithelium, and single cells were inside the follicles.